Methods for measuring self-amplifying RNA and trans amplifying RNA activity in vivo

Abstract

This disclosure provides methods of measuring trans-amplifying RNA and self- amplifying RNA amplification in vivo. The taRNA and the saRNA are barcoded, administered to a subject, and barcode amplification in a specific cell type is measured.

Description

[0001] METHODS FOR MEASURING SELF- AMPLIFYING RNA AND TRANS AMPLIFYING RNA ACTIVITY IN VIVO

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional Application No. 63 / 612,162, filed December 19, 2023, entitled “METHODS FOR MEASURING SELF- AMPLIFYING RNA AND TRANS AMPLIFYING RNA ACTIVITY IN VIVO,” the content of which is hereby incorporated by reference herein in its entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (A141470002WO00-SEQ-ARM.xml; Size: 405,552 bytes; and Date of Creation: December 17, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] RNA therapeutics have revolutionized medicine. Many RNA therapeutics are delivered to a target cell using a nanoparticle (e.g., a lipid nanoparticle). Different nanoparticles can target different cell types, but it is not always clear, based on the nanoparticle’s composition, which cell type will be targeted. Thus, screening methods have been developed to determine the in vivo localization of nanoparticles comprising RNA therapeutics.

[0008] SUMMARY

[0009] A central challenge in treating disease is delivering a therapeutic to a specific cell type of interest. This is particularly important for therapeutics that are typically delivered using a delivery vehicle (e.g., a nanoparticle) like RNA therapeutics. Current methods measure which cell types are targeted by different nanoparticle compositions, which is useful determining in localization of different nanoparticle types. However, these methods do not measure whether the nanoparticle cargo (e.g., the RNA therapeutic) escapes the endosome and reaches the cytoplasm, which is important because many RNA therapeutics have little to no therapeutic effect until they reach the cytoplasm and can be translated or target an endogenous RNA or DNA. More recent methods have sought to measure effective RNA delivery and translation by isolating and measuring barcoded proteins using mass spectrometry or ELISA, but publications of these methods admit unreliable quantitation. For example, Ryhm et al. indicates that peptide- barcode quantitation can be unreliable in certain tissues. Rhym et al., Nature Biomedical

[0010] 12015157.1 Engineering (2023): 1-10. In addition, the sensitivity of peptide barcodes below 0.001 mg / kg protein expression is questionable. Current RNA barcoding methods only measure biodistribution and not in vivo amplification, and biodistribution to cells does not necessary lead to payload expression in those cells.

[0011] In some aspects, provided herein is a method of determining in vivo amplification of a plurality of ribonucleic acid (RNA) polynucleotides in cells of a specific cell type of a subject, the method comprising: (i) obtaining the plurality of RNA polynucleotides, wherein each RNA polynucleotide of the plurality of RNA polynucleotides comprises a conserved sequence element (CSE) and an RNA barcode; (ii) administering RNA polynucleotides of the plurality of RNA polynucleotides to the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein the cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining in vivo amplification of the administered RNA polynucleotides of the plurality of RNA polynucleotides in the subject by detecting each RNA barcode in the extracted cell of the specific cell type.

[0012] In some embodiments, RNA polynucleotides of the plurality of RNA polynucleotides further comprises a nucleic acid encoding a payload.

[0013] In some embodiments, RNA polynucleotides of the plurality of RNA polynucleotides comprise: (a) a 5’ alphavirus untranslated region (UTR); (b) the CSE; (c) a nucleic acid pay load or a nucleic acid encoding a pay load; (d) the RNA barcode; and (e) a 3’ alphavirus UTR. In some embodiments, RNA polynucleotides of the plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ UTR comprising the CSE; (b) a nucleic acid payload or a nucleic acid encoding a payload; (c) the RNA barcode; and (d) a 3’ alphavirus UTR. In some embodiments, RNA polynucleotides of the plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ UTR comprising the CSE; (b) a nucleic acid payload or a nucleic acid encoding a payload; and (c) a 3’ alphavirus UTR comprising the RNA barcode. In some embodiments, the 5’ alphavirus UTR is a Venezuelan Equine Encephalitis Virus (VEEV) 5’-UTR, Semliki Forest Virus (SFV) 5 ’-UTR, Sindbis Virus (SINV) 5 ’-UTR, or Chikungunya Virus (CHIKV) 5 ’-UTR. In some embodiments, the 3’ alphavirus UTR is a VEEV 3 ’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR. In some embodiments, the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR. In some embodiments, the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR. In some embodiments, the RNA barcode is indicative of a mutation in the 5’ alphavirus UTR and / or the 3’ alphavirus UTR of a corresponding RNA polynucleotide.

[0014] 12015157.1 In some embodiments, the RNA barcode comprises at least 6 nucleotides. In some embodiments, the RNA barcode consists of 8 nucleotides. In some embodiments, the RNA barcode comprises the RNA barcode set forth in any one of REF ID NOs. 429-848. In some embodiments, the RNA barcode has minimal to no predicted secondary structure. In some embodiments, the RNA barcode does not comprise a mammalian microRNA (miRNA) seed region or a miRNA target site.

[0015] In some embodiments, the payload comprises a reporter protein. In some embodiments, the reporter protein is green fluorescent protein (GFP), enhanced GFP (EGFP), mCherry, mKate, anchored heavy chain variable domain (aVHH), nano luciferase (nanoLuc), firefly luciferase (Flue), secreted embryonic alkaline phosphatase (SEAP), or Cre recombinase. In some embodiments, the payload is not a reporter protein. In some embodiments, the payload is a therapeutic polynucleotide, or the payload encodes a therapeutic polypeptide. In some embodiments, the payload is a nuclease. In some embodiments, the payload does not encode a protein.

[0016] In some embodiments, each different RNA barcode sequence in the plurality of RNA polynucleotides is at least a Hamming distance of 3 from each other.

[0017] In some embodiments, RNA polynucleotides of the plurality of RNA polynucleotides further comprise nucleic acids encoding the replicase that is cognate to the CSE.

[0018] In some embodiments, the method further comprises contacting, in vivo, cells of the specific cell type of the subject with a polyribonucleotide encoding the replicase that is cognate to the CSE.

[0019] In some embodiments, the plurality of RNA polynucleotides comprises at least 10 different RNA polynucleotides. In some embodiments, the at least 10 different RNA polynucleotides each comprise a different 5’ alphavirus UTRs and / or a different 3’ alphavirus UTRs.

[0020] In some embodiments, obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of nanoparticles, wherein nanoparticles of the plurality of nanoparticles comprise an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, at least one nanoparticle of the plurality of nanoparticles comprises a nucleic acid encoding the replicase that is cognate to the CSE. In some embodiments, contacting cells of the specific cell type comprises contacting, in vivo, the cells of the specific cell type with the plurality of nanoparticles. In some embodiments, obtaining the plurality of nanoparticles

[0021] 12015157.1 comprises obtaining at least two different pluralities of nanoparticles, wherein the different pluralities of nanoparticles comprise a different RNA barcode.

[0022] In some embodiments, nanoparticles of the plurality of nanoparticles are lipid nanoparticles. In some embodiments, the plurality of nanoparticles comprises a first plurality of lipid nanoparticles and a second plurality of lipid nanoparticles, wherein: the first plurality of lipid nanoparticles has a formulation comprising at least one different component relative to the second plurality of lipid nanoparticles, the first plurality of lipid nanoparticles comprises a first plurality of RNA polynucleotides comprising a first RNA barcode and the second plurality of lipid nanoparticles comprises a second plurality of RNA polynucleotides comprising a second RNA barcode, and wherein the first and second RNA barcodes are different.

[0023] In some embodiments, the nanoparticles are polymeric nanoparticles. In some embodiments, the plurality of nanoparticles comprises a first plurality of polymeric nanoparticles and a second plurality of polymeric nanoparticles, wherein: the first plurality of polymeric nanoparticles has a formulation comprising at least one different component relative to the second plurality of polymeric nanoparticles, the first plurality of polymeric nanoparticles comprises a first plurality of RNA polynucleotides comprising a first RNA barcode and the second plurality of polymeric nanoparticles comprises a second plurality of RNA polynucleotides comprising a second RNA barcode, and wherein the first and second RNA barcodes are different.

[0024] In some embodiments, the method further comprises contacting the cells of the specific cell type with a replicase nanoparticle, the replicase nanoparticle (a) comprising a polyribonucleotide encoding the replicase that is cognate to the CSE and (b) not comprising an RNA polynucleotide of the plurality of RNA polynucleotides.

[0025] In some embodiments, a nanoparticle of the plurality of nanoparticles comprises a guide RNA.

[0026] In some embodiments, obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of viral vectors, wherein viral vectors of the plurality of viral vectors comprise an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, at least one viral vector of the plurality of viral vectors comprises a nucleic acid encoding the replicase that is cognate to the CSE. In some embodiments, viral vectors of the plurality of viral vectors comprise adeno-associated viral vectors, lentiviral vectors, or adenoviral vectors. In some embodiments, contacting cells of the specific cell type comprises contacting, in vivo, the cells of the specific cell type with the plurality of viral vectors. In some

[0027] 12015157.1 embodiments, cells of the specific cell type of the subject have been engineered to express the replicase that is cognate to the CSE. In some embodiments, obtaining the plurality of RNA polynucleotides comprises synthesizing the plurality of RNA polynucleotides using in vitro transcription.

[0028] In some embodiments, contacting cells of the specific cell type comprises administering the plurality of RNA polynucleotides to the subject.

[0029] In some embodiments, extracting cells of the specific cell type comprises extracting a biological sample that comprises the specific cell type from the subject. In some embodiments, extracting further comprises isolating cells of the specific cell type from the biological sample using fluorescent assisted or magnetic cell sorting.

[0030] In some embodiments, determining in vivo amplification of the plurality of RNA polynucleotides comprises sequencing the RNA barcodes of the RNA polynucleotides of the plurality of RNA polynucleotides to produce sequencing data. In some embodiments, determining in vivo amplification of a selected RNA polynucleotide of the plurality of RNA polynucleotides in the cells of the specific cell type, comprises detecting the RNA barcode corresponding to the selected RNA polynucleotide in the cells of the specific cell type. In some embodiments, determining in vivo amplification of a selected RNA polynucleotide of the plurality of RNA polynucleotides in the cell of the specific cell type, comprises using the sequencing data to measure an amount of the RNA barcode corresponding to the selected RNA polynucleotide in the cells of the specific cell type. In some embodiments, determining in vivo amplification of the plurality of RNA polynucleotides in the cells of the specific cell type comprises measuring an amount of the RNA barcode in the cells of the specific cell type relative to an amount of the RNA barcode in at least one other cell type of the subject.

[0031] In some aspects, provided herein is a ribonucleic acid (RNA) polynucleotide comprising a 5’ alphavirus untranslated region (UTR), a conserved sequence element (CSE); an RNA barcode comprising at least 6 RNA nucleotides; and a 3’ alphavirus UTR.

[0032] In some embodiments, the 5’ alphavirus UTR comprises the CSE. In some embodiments, the 3’ alphavirus UTR comprises the RNA barcode. In some embodiments, the 5’ alphavirus UTR is a Venezuelan Equine Encephalitis Virus (VEEV) 5’-UTR, Semliki Forest Virus (SFV) 5 ’-UTR, Sindbis Virus (SINV) 5 ’-UTR, or Chikungunya Virus (CHIKV) 5 ’-UTR. In some embodiments, the 3’ alphavirus UTR is a VEEV 3 ’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR. In some embodiments, the 5’ alphavirus UTR comprises a mutation relative to

[0033] 12015157.1 a wildtype 5’ alphavirus UTR. In some embodiments, the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR.

[0034] In some embodiments, the RNA barcode is indicative of mutation in the 5’ alphavirus UTR and / or the 3’ alphavirus UTR.

[0035] In some embodiments, each barcode has minimal to no predicted secondary structure. In some embodiments, each barcode does not comprise a mammalian microRNA (miRNA) seed region or target site. In some embodiments, the RNA barcode consists of 8 nucleotides. In some embodiments, the RNA barcode is the RNA barcode set forth in any one of REF ID NOs.: 429- 848.

[0036] In some embodiments, the RNA polynucleotide comprises the sequence set forth in any one of SEQ ID NOs: 9-428.

[0037] In some embodiments, the RNA polynucleotide further comprises a nucleic acid payload or a nucleic acid encoding a payload. In some embodiments, the payload comprises a reporter protein. In some embodiments, the reporter protein is green fluorescent protein (GFP), enhanced GFP (EGFP), mCherry, mKate, anchored heavy chain variable domain (aVHH), nano luciferase (nanoLuc), firefly luciferase (Flue), Secreted embryonic alkaline phosphatase (SEAP), or Cre recombinase. In some embodiments, the payload is not a reporter protein. In some embodiments, the payload is a therapeutic polynucleotide, or the payload encodes a therapeutic polypeptide. In some embodiments, the payload is a nuclease. In some embodiments, the payload does not encode a protein.

[0038] In some embodiments, the RNA polynucleotide further comprises a nucleic acid encoding a replicase that is cognate to the CSE.

[0039] In some aspects, provided herein is a trans-amplifying RNA comprising: (a) a first RNA polynucleotide comprising an RNA polynucleotide described herein; and (b) a second RNA polynucleotide comprising a nucleic acid encoding a replicase that is cognate to the CSE.

[0040] In some aspects, provided herein is a composition comprising a plurality of nanoparticles, wherein each nanoparticle of the plurality comprises an RNA polynucleotide described herein or a taRNA described herein.

[0041] In some aspects, provided herein is a composition comprising a plurality of nanoparticles and a plurality of trans-amplifying ribonucleic acids (RNA) (taRNAs), wherein taRNAs of the plurality of taRNAs comprise: a first RNA polynucleotide comprising a conserved sequence element (CSE) and an RNA barcode; and a second RNA polynucleotide comprising a nucleic

[0042] 12015157.1 acid encoding a replicase that is cognate to the CSE; wherein the plurality of taRNAs comprises a first taRNA and second taRNA, wherein the first taRNA comprises a different RNA barcode relative to the second taRNA; and wherein the plurality of nanoparticles comprises a first plurality of nanoparticles and a second plurality of nanoparticles, wherein nanoparticles of the first plurality of nanoparticles comprises the first taRNA and do not comprise the second taRNA; and wherein nanoparticles of the second plurality of nanoparticles comprise the second taRNA and do not comprise the first taRNA.

[0043] In some aspects, provided herein is a composition comprising a plurality of nanoparticles and a plurality of self-amplifying ribonucleic acids (RNA) (saRNAs), wherein saRNAs of the plurality of saRNAs comprise an RNA polynucleotide comprising a conserved sequence element (CSE), an RNA barcode, and a nucleic acid encoding a replicase that is cognate to the CSE; wherein the plurality of saRNAs comprises a first saRNA and second saRNA, wherein the first saRNA comprises a different RNA barcode relative to the second saRNA; and wherein the plurality of nanoparticles comprises a first plurality of nanoparticles and a second plurality of nanoparticles, wherein nanoparticles of the first plurality of nanoparticles comprises the first saRNA and do not comprise the second saRNA; and wherein nanoparticles of the second plurality of nanoparticles comprise the second saRNA and do not comprise the first saRNA.

[0044] In some embodiments, the first plurality of nanoparticles has a different formulation relative to the second plurality of nanoparticles.

[0045] In some aspects, provided herein is a method of determining in vivo amplification of a plurality of RNA polynucleotides in a specific cell type of a subject, the method comprising: (i) obtaining plurality of nanoparticles collectively comprising a plurality of RNA polynucleotides, wherein each RNA polynucleotide of the plurality of RNA polynucleotides comprises a conserved sequence element (CSE) and an RNA barcode; (ii) contacting, in vivo, nanoparticles of the plurality of nanoparticles with cells of the specific cell type of the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining in vivo amplification of the plurality of RNA polynucleotides in the subject by measuring an amount of each RNA barcode in the extracted cells of the specific cell type.

[0046] In some aspects, provided herein is a method of determining delivery of a plurality of RNA polynucleotides to the cytosol of a specific cell type of a subject, the method comprising: (i) obtaining a plurality of nanoparticles collectively comprising a plurality of RNA polynucleotides, wherein each RNA polynucleotide of the plurality of RNA polynucleotides

[0047] 12015157.1 comprises a conserved sequence element (CSE) and an RNA barcode; (ii) contacting, in vivo, nanoparticles of the plurality of nanoparticles with cells of the specific cell type of the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining delivery of the plurality of RNA polynucleotides to the cytosol of the specific cell type by measuring an amount of RNA barcode produced by in vivo amplification in the cytosol of the extracted cells of the specific cell type.

[0048] In some embodiments, the plurality of nanoparticles comprises lipid nanoparticles.

[0049] In some aspects, provided herein is a method of preparing a composition for delivery of ribonucleic acid (RNA) cargo to the cytosol of a cell of a specific cell type in a subject, the method comprising: (a) obtaining a first plurality of nanoparticles and a second plurality of nanoparticles, wherein: the first plurality of nanoparticles has a different formulation relative to the second plurality of nanoparticles, the first plurality of nanoparticles comprises a first RNA polynucleotide comprising a first RNA barcode and the second plurality of nanoparticles comprises a second RNA polynucleotide comprising a second RNA barcode, and wherein the first and second RNA barcodes are different; (b) administering the first and second pluralities of nanoparticles to the subject; (c) determining that at least one of the first and / or second RNA polynucleotides was delivered to the cytosol of cells of the specific cell type by identifying the RNA barcode present in the cytosol of the cells of the specific cell type; and (d) preparing a composition comprising a plurality of nanoparticles comprising RNA cargo and having the formulation of the nanoparticle comprising the RNA barcode identified as delivered to the cytosol of the cells of the specific cell type in step (c).

[0050] In some embodiments, the first plurality of nanoparticles and the second plurality of nanoparticles are a first plurality of lipid nanoparticles and a second plurality of lipid nanoparticles.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 shows the difference between lipid nanoparticle (LNP) localization to a specific cell type and delivery of an LNP (LNP D) to the cytosol. While LNPs A-D are all distributed to the cell, only LNP D is functional for delivery, as LNPs A-C all terminate outside the cytosol. Despite only LNP D being functional for delivery, existing nanoparticle barcoding systems would have read LNPs A-D out as equally potent.

[0053] 12015157.1 FIGs. 2A-2B show a barcoded trRNA and trRNA amplification in vitro by a replicase. FIG. 2A shows a diagram of a trRNA with the GFP payload and a barcode. FIG. 2A shows amplification of the trRNA in cells with and without replicase addition.

[0054] FIGs. 3A-3C show a positive correlation between the amount of trRNA (11 bp- ampRNAs) transfected into a cell and the amount of trRNA measured after amplification by the replicase. The 1 Ibp ampRNAs have different barcodes and were transfected in different amounts to the cells. FIG 3A shows GFP+ positive cells 24 hour after transfection. FIG. 3B shows there is nearly 66-fold more GFP signal in cells that received the replicase than cells that did not receive the replicase. FIG. 3C shows a strong correlation between isolated RNA (+ / - replicase) to input library.

[0055] FIG. 4 is a diagram of a method for screening different LNP types in vivo for delivery of taRNA to the cytosol and trRNA amplification. In this method, each LNP comprises a trRNA and a replicase construct. Different LNPs types have different trRNA barcodes.

[0056] FIG. 5 is a diagram of a method for screening different LNP types in vivo for delivery of a trRNA to the cytosol and trRNA amplification. In this method different LNP types comprise trRNAs with different barcodes, but do not also comprise a replicase construct. Rather, a second LNP comprises the replicase construct. The LNPs comprising the trRNAs and the LNPs comprising the replicase construct are both administered to the subject.

[0057] FIG. 6 illustrates a novel nanoparticle barcoding system where a barcode sequence is inserted into the trRNA, which amplifies in the cytosol in the presence of replicase protein. In one embodiment of the screening system, multiple (N) lipid nanoparticles are formulated such that each carry a replicase mRNA and a uniquely barcoded trRNA, such that each LNP carries the same type of replicase mRNA and a barcoded trRNA with a unique sequence.

[0058] FIGs. 7A-7B illustrate the proof of concept for a barcoded taRNA screen. In FIG. 7A, mice were administered intramuscularly with barcoded mRNA (an existing method for LNP screening) and barcoded taRNA. The abundance of barcoded RNA was quantified over a time course via qPCR. In FIG. 7B, cells were transfected with an intentional varying amount of barcodes trRNA and relative abundance of each barcode was measured via NGS before and after co-transfection with a replicase into mammalian cells.

[0059] FIG. 8 illustrates an exemplary nanoparticle screening workflow wherein multiple nanoparticles are formulated to carry barcoded taRNA, each NP then individually undergoes various methods of characterization, and multiple NPs are pooled and administered into one or multiple species. After a certain timepoint, tissues and (optionally) specific cell-types are

[0060] 12015157.1 isolated and the barcoded taRNA is then measured by NGS. These NGS results are then related to the NP formulation (e.g. components & ratio of components) and characterization results. These data may inform hit / lead nomination or inform iterative screening efforts.

[0061] FIGs. 9A-9C are screening data wherein 8 LNP compositions were formulated into formulation duplicates to generate 16 LNPs, and then administered into 8 mice. FIG. 9A shows sources of variability based on technical or biological differences for the 16 LNPs. Two exemplary field-known LNPs are assessed in FIGs. 9B-9C.

[0062] FIGs. 10A-10B show the correlation between barcode results (enrichment ratio) and the performance of each LNP individually (measured via IVIS) for a test pool of 5 compositions after intramuscular administration (FIG. 10A) and intravenous administration to liver expression (FIG. 10B).

[0063] FIGs. 11A-11B show the impact of the addition of GalNAc targeting ligands to three LNP compositions in the liver (FIG. 11A) and spleen (FIG. 11B).

[0064] FIG. 12 shows expression of barcoded trRNAs in BHK-21 cells at 24 hours.

[0065] DETAILED DESCRIPTION

[0066] In some aspects, this disclosure provides compositions and methods useful for, in some embodiments, determining in vivo amplification of ribonucleic acid (RNA) cargo to the cytosol of a specific cell type of a subject (e.g., by a nanoparticle). In some aspects, this disclosure provides methods useful for determining a nanoparticle is suitable for delivering RNA cargo to the cytosol of a specific cell type of a subject.

[0067] Amplifying RNA

[0068] In some aspects, this disclosure provides method of determining in vivo amplification of a ribonucleic acid (RNA) polynucleotide in a specific cell type of a subject.

[0069] “Zn vivo” refers to taking place in a multi-cellular organism. Multi-cellular organisms include but are not limited to, animals, plants, multi-cellular fungi, and multi-cellular algae. In some embodiments, in vivo refers to an event (e.g., amplification of an RNA) taking place in a mammal. In some embodiments, in vivo refers to an event (e.g., amplification of an RNA) taking place in a mouse or rat. In some embodiments, in vivo refers to an event (e.g., amplification of an RNA) taking place in a human.

[0070] A “polynucleotide” refers to a polymer of nucleotides. A polynucleotide is generally composed of nucleotides that are naturally found in DNA or RNA (e.g., adenosine / deoxyadenosine (A), thymidine / deoxythymidine (T), guano sine / deoxy guano sine (G),

[0071] 12015157.1 cytidine / deoxycytidine (C) and uridine (U) joined by phosphodiester bonds. However, the term “polynucleotide” may also refer to polynucleotides comprising nucleotides or nucleotide analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications.

[0072] “Amplification” refers to production of one or more copies of a template IRNA polynucleotide by a cell (e.g., a cell containing a trans replicating RNA (trRNA) and / or a selfamplifying RNA). A “template” RNA polynucleotide refers to any RNA polynucleotide used by a polymerase (e.g., a replicase) to make “copies” of the RNA polynucleotides. In some embodiments, a template RNA polynucleotide is not a copy of a template RNA polynucleotide. In some embodiments, a template RNA polynucleotide is a copy of another template RNA polynucleotide. In some embodiments, amplification refers to producing a plurality of copies of a template RNA polynucleotide at a sufficient amount such that the copies of the RNA polynucleotide are detectable (e.g., via quantitative PCR (qPCR) or next-generation sequencing). In some embodiments, each copy of a plurality of copies of the template RNA polynucleotide is 100% identical to the template RNA polynucleotide. In some embodiments, one or more copies of a plurality of copies of a template RNA polynucleotide are not 100% identical to the template RNA polynucleotide. For example, a replicase producing copies of a template RNA polynucleotide by amplifying the template RNA polynucleotide may introduce a mutation into a copy of the template RNA polynucleotide.

[0073] In some embodiments, an RNA polynucleotide (e.g., a template RNA polynucleotide) is amplified by a replicase. A “replicase” is an RNA-dependent RNA polymerase capable of transcribing (i.e., reading) a template RNA (e.g., a trRNA) to produce a copy. In some embodiments, the replicase is an alphavirus replicase. The term “alphavirus” refers to an RNA virus belonging to the Togaviridae family. In some embodiments, the alphavirus comprises a single- stranded RNA genome encoding at least nsPl, nsP2, nsP3, nsP4, El, E2, E3, 6K / TF and capsid proteins. An alphavirus may be any alphavirus known in the art; non-limiting examples include Venezuelan equine encephalitis virus (VEEV), Semliki Forest virus (SFV), Sindbis virus SINV), and Chikungunya virus (CHIKV).

[0074] Typically, an alphaviral replicase comprises a complex formed by the non-structural proteins nsPl, nsP2, nsP3, and nsP4. Once expressed, alphaviral replicase may interact with a RNA polynucleotide comprising one or more conserved sequence elements (CSEs) and generate mirrored copies of it, which can be subsequently translated. A “conserved sequence element

[0075] 12015157.1 (CSE),” refers to a recognition site for a cognate alphavirus replicase. “Cognate” as used herein refers to a CSE - replicase pairing that is sufficient to result in replicase driven amplification of at least a portion of the polynucleotide comprising the CSE. For example, a CSE and a replicase that are from the same alphavirus species are cognate. A replicase that is cognate to a CSE is capable of transcribing a portion of a polynucleotide comprising the CSE. In some embodiments, a replicase and a CSE from the same species (e.g., alphavirus) are cognate. In some embodiments, a replicase and CSE from different alphaviruses are cognate.

[0076] Typically, a CSE functions as a core promoter or enhancer for initiation of replication of a downstream sequence, such that a 5 ’-CSE may initiate synthesis of a plus-strand and a 3 ’-CSE may initiate synthesis of a minus-strand. An RNA polynucleotide may comprise one or more 5’- CSEs and / or 3’-CSEs. In some embodiments, a CSE forms one or more secondary structure(s), such as one or more stem-loops. Non-limiting examples of CSEs include CSE1, CSE2, CSE3, CSE4, and variants or derivatives thereof. In some embodiments, the CSE is a CSE from VEEV, SFV, SINV, or CHIKV alphavirus. CSEs are known in the art, e.g., as described in Hyde JL, Virus Res. 2015 Aug 3;206:99-107.

[0077] Trans-amplifying and Self-amplifying RNAs

[0078] In some embodiments, an RNA polynucleotide (e.g., a template RNA polynucleotide) comprises a CSE. In some embodiments, an RNA polynucleotide comprises a CSE and a nucleic acid encoding a replicase (e.g., a cognate replicase). In some embodiments, an RNA polynucleotide is a self-amplifying RNA (saRNA). An saRNA is an RNA polynucleotide comprising a CSE and a nucleic acid encoding a replicase in the same molecule. An external source of a replicase (e.g., a nucleic acid encoding a replicase) is not typically required for amplification of the saRNA. saRNAs are known, e.g., as described in Comes JDG et al., Trends Biotechnol. 2023 Nov;41(l l): 1417-1429.

[0079] In some embodiments, an RNA polynucleotide is a trans replicating RNA (trRNA). A trRNA is an RNA polynucleotide comprising a CSE and not comprising a nucleic acid encoding a replicase. To amplify a trRNA, a replicase is provided by another source (e.g., by a cell, or by a separate RNA polynucleotide encoding the replicase). A trans-amplifying RNA (taRNA) refers to a trRNA and a separate RNA encoding a replicase (i.e., a replicase construct).

[0080] A “replicase construct” is a polyribonucleic acid comprising nucleic acids encoding a replicase from an RNA virus, for example, an alphavirus. In some embodiments, the replicase of the replicase construct is modified (e.g., to comprise one or more mutations). A replicase

[0081] 12015157.1 construct may be comprised in a taRNA (e.g., as an RNA polynucleotide not comprising a CSE). An saRNA comprises a replicase construct.

[0082] In some embodiments, a replicase construct encodes a replicase derived from a Semliki forest virus (SFV) (SFV replicase). In some embodiments, a replicase construct encodes a replicase derived from a Sindbis virus (SINV) (SINV replicase).

[0083] Determining in vivo amplification of an RNA polynucleotide or a plurality of RNA polynucleotides can be performed using any suitable method (e.g., using qPCR, next generation sequencing, or northern blot). In some embodiments, determining in vivo amplification of an RNA polynucleotides (e.g., of a template RNA polynucleotide) comprises performing nextgeneration sequencing to sequence the RNA polynucleotides (e.g., the template RNA polynucleotides) and copies of the plurality of RNA polynucleotides. In some embodiments, determining in vivo amplification of a plurality of RNA polynucleotides comprises performing next-generation sequencing to sequence the plurality of RNA polynucleotides and copies of the plurality of RNA polynucleotides. In some embodiments, the next- generation sequencing comprising sequencing using IFFUMINA, SOFID, PACBIO, Nanopore or ION TORRENT sequencing.

[0084] In some embodiments, determining in vivo amplification of an RNA polynucleotide comprises using sequencing data from the sequencing to determine an abundance of each RNA polynucleotide of a plurality of RNA polynucleotides. In some embodiments, determining in vivo amplification comprises using sequencing data from the sequencing to determine enrichment of a specific RNA polynucleotide from among a plurality of different RNA polynucleotides. In some embodiments, determining in vivo amplification comprises comparing the amount (e.g., number of copies) of a given RNA barcode present in a cell in a subject before administration of an RNA polynucleotide comprising the RNA barcode to the subject to the amount (e.g., number of copies) of the given RNA barcode in the cell in the subject after administration of the RNA polynucleotide comprising the RNA barcode to the subject, e.g., after extraction from the subject (e.g., as a ratio).

[0085] RNA Barcodes

[0086] In some aspects, this disclosure provides RNA barcodes useful for determining delivery of RNA cargo to the cytosol of a cell. In some embodiments, this disclosure provides an RNA polynucleotide comprising an RNA barcode.

[0087] 12015157.1 An “RNA barcode” refers to a non-coding (e.g., not encoding a protein, not encoding a nucleic acid pay load) and non-functional (e.g., not functioning as a promoter or terminator) portion of an RNA polynucleotide that can be used to identify the RNA polynucleotide.

[0088] In some embodiments, an RNA barcode comprises at least 6 nucleotides (at least 6 nucleotides, at least 8 nucleotides, at least 10 nucleotides, at least 15 nucleotides, or at least 20 nucleotides). In some embodiments, an RNA barcode comprises 6-20 nucleotides. In some embodiments, an RNA barcode comprises 8-20 nucleotides. In some embodiments, an RNA barcode comprises 8 nucleotides. In some embodiments, an RNA barcode comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, an RNA barcode is randomly determined or generated.

[0089] In some embodiments, different RNA barcodes comprise sequence differences measurable by an edit distance. Several measures of edit distance are known in the art, for example, Hamming distance and Levenshtein distance (e.g., see Berger, Bonnie, Michael S. Waterman, and Yun William Yu. "Levenshtein distance, sequence comparison and biological database search." IEEE transactions on information theory 67.6 (2020): 3287-3294.). In some embodiments, an RNA barcode is a Levenshtein distance of at least 2 from each other barcode of a plurality of RNA barcodes. In some embodiments, an RNA barcode is a Levenshtein distance of at least 3 from each other barcode of a plurality of RNA barcodes. In some embodiments, each RNA barcode of a plurality of RNA barcodes is a Levenshtein distance of at least 2 (e.g., at least 3) from each other RNA barcode in a plurality of RNA barcodes. In some embodiments, an RNA barcode is a Hamming distance of at least 2 from each other barcode of a plurality of RNA barcodes. In some embodiments, an RNA barcode is a Hamming distance of at least 3 from each other barcode of a plurality of RNA barcodes. In some embodiments, each RNA barcode of a plurality of RNA barcodes is a Hamming distance of at least 2 (e.g., at least 3) from each other RNA barcode in the plurality.

[0090] In some embodiments, an RNA barcode is comprised in consecutive nucleotides (i.e., each nucleotide in an RNA barcode is consecutive). In some embodiments, the RNA barcode is not comprised in consecutive nucleic acids. For example, a non-consecutive 6-nucleotide RNA barcode may be comprised across 12 nucleotides, such that every other nucleotide of the RNA barcode is interspersed across 12 nucleotides (e.g., every other nucleotide of the 12 nucleotides is a nucleotide of the 6-nt RNA barcode).

[0091] In some embodiments, the RNA barcode has minimal to no predicted secondary structure. Secondary structure of RNA barcodes may be predictable using any suitable

[0092] 12015157.1 algorithm. For example, secondary structure may be determined using a method described in Zhao Q et al., PLoS Comput Biol. 2021 Aug 26;17(8):el009291. In some embodiments, secondary structure is determined using ViennaRNA e.g., as described in Lorenz, R. et al., ViennaRNA Package 2.0. Algorithms Mol Biol 6, 26 (2011). In some embodiments, minimal to no predicted secondary structure of RNA barcode is determined by inputting potential RNA barcode sequences into ViennaRNA and then selecting RNA barcodes with minimal predicted deltaG. In some embodiments, an RNA barcode, in combination with the nucleotides on either side of the RNA barcode (e.g., 5’ and 3’ 10-20 nucleotides) have little to no secondary structure. In some embodiments, an RNA barcode has no secondary structure (e.g., does not form a stem loop under physiological conditions).

[0093] In some embodiments, an RNA barcode does not comprise a microRNA (miRNA) seed region. In some embodiments, an RNA barcode does not comprise a mammalian miRNA seed region. miRNA seed regions are well known, e.g., as described in Vikram Agarwal et al., (2015) eLife 4:e05005. In some embodiments, an RNA barcode does not comprise or form a part of a binding site for a miRNA, e.g., as provided by Y. Chen et al., (2020) “miRDB: an online database for prediction of functional microRNA targets.” Nucleic Acids Research. 48(D1):D127-D131.

[0094] In some embodiments, an RNA barcode identifies a unique RNA polynucleotide of a plurality of RNA polynucleotides. For example, each RNA polynucleotide in a plurality of RNA polynucleotides may comprise a different RNA barcode, such that measuring the amount of a given RNA barcode can be used to determine the amount of the corresponding RNA polynucleotide in which the given RNA barcode was comprised. In another example, each RNA polynucleotide in a plurality of RNA polynucleotides may comprise a different RNA barcode, such that the amplification of (e.g., the number of copies of) a template RNA polynucleotide can be determined by measuring the amount of the RNA barcode comprised in the template RNA polynucleotide.

[0095] In some embodiments, an RNA barcode identifies a mutation (e.g., substitution of a first nucleotide for another, deletion, and / or insertion) in an RNA polynucleotide in which the RNA barcode is comprised. For example, each RNA polynucleotide in a plurality of RNA polynucleotides may comprise a different mutation relative to another RNA polynucleotide; these different mutations may be present in any position of an RNA polynucleotide, making them difficult and / or expensive to identify (e.g., using sequencing) with increasing numbers of unique RNA. To improve identification, unique RNA barcodes may be incorporated into each

[0096] 12015157.1 different RNA polynucleotide, such that each RNA barcodes is indicative of a mutation in the RNA polynucleotide in which it is comprised. The RNA barcode may then be sequenced as a proxy for sequencing the mutation. Preparation for sequencing may include reverse transcribing the RNA barcode into a DNA barcode the sequencing the DNA barcode.

[0097] In some embodiments, an RNA barcode comprises a sequence corresponding to any one of REF ID NOs: 429-848. In some embodiments, an RNA barcode consists of the sequence set forth in any one of REF ID NOs: 428-848.

[0098] An RNA barcode may be in any suitable location in an RNA polynucleotide. Suitable locations may be determined by, for example, identifying a location in which insertion of the RNA barcode would have minimal to no effect on RNA polynucleotide amplification compared to an otherwise identical RNA polynucleotide lacking the RNA barcode). In some embodiments, inserting an RNA barcode into an RNA polynucleotide reduces amplification of the RNA polynucleotide by no more than 40% (e.g., no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, or no more than 0.1%) relative to the RNA polynucleotide without the inserted barcode. In some embodiments, inserting an RNA barcode into an RNA polynucleotide increases amplification of the RNA polynucleotide by no more than 40% (e.g., no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, or no more than 0.1%) relative to the RNA polynucleotide without the inserted barcode.

[0099] Untranslated Regions

[0100] In some embodiments, an RNA polynucleotide comprises one or more untranslated regions. An “untranslated region,” hereinafter referred to as “UTR,” is a region in a polynucleotide which may be transcribed, but is not translated into a gene product. UTRs may act as stabilizing elements and / or provide regulation of transcription of a gene or transgene. Typically, UTRs are found upstream and / or downstream of a gene or transgene. A UTR located directly upstream of a start codon operably linked to a gene or transgene is referred to herein as a

[0101] 12015157.1 5’-UTR. As a skilled artisan will understand, 5’-UTRs may comprise sequence elements which play roles in regulation of expression (e.g., Kozak sequences) or structural elements which alter stability of the molecule (e.g., 5’ cap structures). A UTR located directly downstream of a stop codon operably linked to a gene or transgene is referred to herein as a 3 ’-UTR. 3’-UTRs may comprise structural elements which alter the stability of a construct and / or provide transcriptional control, including, but not limited to AU-rich elements and polyA tails. A variety of 5’-UTRs and a 3’-UTRs are known to those of ordinary skill in the art. UTRs may be naturally occurring or synthetic. In some embodiments, the RNA polynucleotide comprises a 5’- UTR and / or a 3 ’-UTR. In some embodiments, a trRNA comprises one or more CSEs, wherein the CSEs are present in one or more UTRs.

[0102] In some embodiments, an RNA polynucleotide comprises a 5 ’-UTR of an alphavirus (e.g., an alphavirus 5’-UTR). In some embodiments, the RNA polynucleotide comprises a 3’- UTR of an alphavirus (e.g., an alphavirus 3’-UTR). Alphavirus 5’-UTR and alphavirus 3’-UTR sequences are described in the art, e.g., by Hyde JL et al., Virus Res. 2015 Aug 3;206:99-107. In some embodiments, the alphavirus 5’-UTR is a Venezuelan equine encephalitis virus (VEEV) 5’-UTR (i.e., a “5’-VEEV-UTR), Semliki Forest Virus (SFV) 5’-UTR (i.e., a “5’-SFV-UTR), Sindbis Virus (SINV) 5 ’-UTR (i.e., a “5’-SINV-UTR), or Chikungunya Virus (CHIKV) 5 ’-UTR (i.e., a “5’-CHIKV-UTR). In some embodiments, the alphavirus 3’-UTR is a 3’-VEEV-UTR, 3’-SFV-UTR, 3’-SINV-UTR, or 3’-CHIKV-UTR.

[0103] In some embodiments, the RNA polynucleotide comprises a 5 ’-UTR and 3 ’-UTR from the same virus. In some embodiments, the RNA polynucleotide comprises a 5’-VEEV-UTR and / or 3’-VEEV-UTR. In some embodiments, the RNA polynucleotide comprises a 5’-SFV- UTR and / or 3’-SFV-UTR. In some embodiments, the RNA polynucleotide comprises a 5’- SINV-UTR and / or 3’-SINV-UTR. In some embodiments, the RNA polynucleotide comprises a 5’-CHIKV-UTR and / or 3’-CHIKV-UTR. In some embodiments, the RNA polynucleotide comprises a 5 ’-UTR and 3 ’-UTR from different alphaviruses; for example, an RNA polynucleotide may comprise a 5’-SFV-UTR and a 3’-SINV-UTR.

[0104] In some embodiments, an alphavirus 5 ’-UTR comprises one or more mutations relative to a wildtype alphavirus 5 ’-UTR. In some embodiments, an alphavirus 3 ’-UTR comprises one or more mutations relative to a wildtype alphavirus 3 ’-UTR.

[0105] In some embodiments, an alphavirus 5 ’-UTR comprises the CSE.

[0106] In some embodiments, a replicase construct comprises a 5 ’-UTR derived from human alpha-globin (5’-HBA-UTR). An exemplary 5’-HBA-UTR is provided in SEQ ID NO: 5. In

[0107] 12015157.1 some embodiments, a replicase construct comprises a 3’-UTR derived from human alpha- globin (3’-HBA-UTR). An exemplary 5’-HBA-UTR is provided in SEQ ID NO: 6. In some embodiments, a replicase construct comprises a 5’-HBA-UTR and a 3’-HBA-UTR. In some embodiments, a replicase construct comprises a 5’-HBA-UTR, a Semliki Forest Virus replicase- encoding sequence, and a 3’-HBA-UTR (5’-HBA-UTR-SFV replicase-3’-HBA-UTR). An exemplary 5’-HBA-UTR-SFV replicase-3’-HBA-UTR construct is provided in SEQ ID NO: 1.

[0108] In some embodiments, an RNA barcode is located in a 3’-UTR of an RNA polynucleotide. In some embodiments, an RNA barcode is located in an alphaviral 3’-UTR of the RNA polynucleotide. In some embodiments, an RNA barcode is located in a SINV 3’-UTR of an RNA polynucleotide. In some embodiments, an RNA barcode is located in the following sequence context of a SINV 3’-UTR: CTCTACAAATGATAATAG-RNA barcode- TCGAGGCGGCCGCCACGC (SEQ ID NO: 429). The skilled artisan will appreciate that the recitation of an “RNA barcode” in SEQ ID NO: 429 represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length. In some embodiments, SEQ ID NO: 429represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length and otherwise conforming to the definition of “RNA barcode” as used throughout the instant disclosure. Illustrative examples of 8nt RNA barcodes (e.g., REF ID NOs: 429-848) in context of SINV 3’ UTRs are provided in SEQ ID NOs: 9-428 (see: Table 2); the skilled artisan will also appreciate that RNA barcodes may be similarly placed in suitable alphavirus UTRs (e.g., as described herein).

[0109] Payload

[0110] In some embodiments, an RNA polynucleotide (e.g., an RNA polynucleotide comprising a barcode) comprises a nucleic acid payload or a nucleic acid encoding a payload. A “payload” refers to one or more gene products of interest for delivery to or expression by an organism. A pay load may be a functional nucleic acid (e.g., RNA), a protein, a peptide or protein fragment, or a fusion protein. In some embodiments, an RNA polynucleotide comprises a CSE and a nucleic acid pay load or a nucleic acid encoding a pay load; The simultaneous expression of a replicase and presence of an RNA polynucleotide comprising a CSE and a nucleic acid payload or a nucleic acid encoding a payload in a cell can thus result in amplification of the RNA polynucleotide and its encoded payload.

[0111] In some embodiments, a payload is a selectable marker. As used herein, a “selectable marker” is a peptide or protein that can be used to screen cells by artificial selection. Nonlimiting examples of selectable markers include antibiotic resistance proteins (e.g., ampicillin, puromycin) and negative selection markers (e.g., thymidine kinase). In some embodiments, a

[0112] 12015157.1 payload is a reporter. A “reporter” is a peptide or protein which alters the appearance of a cell such that cells can be visually or optically screened for presence or absence of the peptide or protein. In some embodiments, a reporter is an enzyme which alters the appearance of a cell, such as beta-galactosidase. In some embodiments, a reporter is a peptide or peptide fragment (e.g., secreted embryonic alkaline phosphatase (SEAP)) which can be detected in combination with additional reagents (e.g., as say- specific media). In some embodiments, a reporter is a fluorophore, such as, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), or any derivative thereof. In some embodiments, a reporter is a recombinase, such as a Flp recombinase, Cre recombinase, or derivative thereof.

[0113] In some embodiments, the payload is “therapeutic payload,” here referring to a gene product useful for treating or preventing a disease or disorder. In some embodiments, a therapeutic payload knocks down, knocks in, increases, inhibits, or otherwise modulates gene expression. In some embodiments, a therapeutic payload replaces or edits an endogenous gene or gene product. In some embodiments, the payload does not encode a protein. In some embodiments, a therapeutic payload is a functional RNA; non-limiting examples of include short hairpin RNA (shRNA), microRNA (miRNA), artificial microRNA (amiRNA), small interfering RNA (siRNA), or circular RNA (circRNA). In some embodiments, a therapeutic pay load is a protein. A therapeutic protein may replace or interfere with activity of deficient or absent endogenous proteins; augment activity of existing metabolic or synthetic pathways; provide a novel function or activity; or interfere with the activity of a pathogen or toxic molecule. Nonlimiting examples of therapeutic proteins suitable for use as payloads include membrane proteins, membrane-associated proteins, secreted proteins, intracellular proteins, immunomodulatory proteins, antigens, antibodies, or fragments thereof.

[0114] In some embodiments, the payload is a nuclease. In some embodiments, the payload is a genome editing enzyme. In some embodiments, the genome editing enzyme comprises a CRISPR associated enzyme (Cas). In some embodiments, the Cas protein is from a CRISPR type I system, a CRISPR type II system, or a CRISPR type III system. In some embodiments, the Cas protein is an RNA cleaving Cas protein. In some embodiments, the Cas protein is an DNA cleaving Cas protein. In some embodiments, the Cas protein is a nickase. In some embodiments, the Cas protein is a Cas9, Cas 10, Casl 1 or Cas 12 protein. In some embodiments, the genome editing enzyme is a base editor (e.g., a cytosine base editor or an adenosine base editor). In some embodiments, the genome editing enzyme is a prime editor. In some

[0115] 12015157.1 embodiments, the genome editing enzyme is a Talen protein. In some embodiments, the pay load encodes an enzyme and comprises nucleic acids, that when transcribed, produces a corresponding guide RNA (e.g., a CRISPR guide RNA, a prime editing RNA) for a therapeutic target gene or transcript (e.g., targeting an oncogene).

[0116] Exemplary RNA polynucleotides

[0117] In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR; (b) a CSE; (c) an RNA barcode; and (d) a 3’ alphavirus UTR. In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR comprising a CSE; (b) an RNA barcode; and (c) a 3’ alphavirus UTR. In some embodiments, an RNA polynucleotide comprises, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) an RNA barcode; and (c) a 3’ alphavirus UTR. In some embodiments, RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5' alphavirus UTR comprising a CSE; (b) an RNA barcode; and (c) a 3’ alphavirus UTR. In some embodiments, most RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5' alphavirus UTR comprising a CSE; (b) an RNA barcode; and (c) a 3’ alphavirus UTR. In some embodiments, each RNA polynucleotide of a plurality of RNA polynucleotides comprises, from 5’ to 3’: (a) a 5' alphavirus UTR comprising a CSE; (b) an RNA barcode; and (c) a 3’ alphavirus UTR.

[0118] In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR; (b) a CSE; and (c) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, an RNA polynucleotide comprises, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5' alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, most RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5' alphavirus UTR comprising a CSE; (b) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, each RNA polynucleotide of a plurality of RNA polynucleotides comprises, from 5’ to 3’: (a) a 5' alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA barcode.

[0119] In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR; (b) a CSE; (c) an RNA barcode; and (d) a 3’ alphavirus UTR; and does not comprise a nucleic acid payload or a nucleic acid encoding a payload; such RNA polynucleotides are also referred to herein as “amp trRNA”. In some embodiments, an RNA polynucleotide consists of, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA

[0120] 12015157.1 barcode. In some embodiments, RNA polynucleotides of a plurality of RNA polynucleotides consist of, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, most RNA polynucleotides of a plurality of RNA polynucleotides consist of, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, each RNA polynucleotide of a plurality of RNA polynucleotides consists of, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; and (b) a 3’ alphavirus UTR comprising an RNA barcode.

[0121] In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR; (b) a CSE; (c) a nucleic acid payload or a nucleic acid encoding a payload; (d) an RNA barcode; and (e) a 3’ alphavirus UTR. In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid payload or a nucleic acid encoding a pay load; (c) an RNA barcode; and (d) a 3’ alphavirus UTR. In some embodiments, RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid payload or a nucleic acid encoding a payload; (c) an RNA barcode; and (d) a 3’ alphavirus UTR. In some embodiments, most of the RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid payload or a nucleic acid encoding a payload; (c) an RNA barcode; and (d) a 3’ alphavirus UTR. In some embodiments, each of the RNA polynucleotides of the plurality of RNA polynucleotides comprises, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid payload or a nucleic acid encoding a pay load; (c) an RNA barcode; and (d) a 3’ alphavirus UTR.

[0122] In some embodiments, an RNA polynucleotide comprises (a) a 5’ alphavirus UTR; (b) a nucleic acid pay load or a nucleic acid encoding a pay load; and (c) a 3’ alphavirus UTR comprising an RNA barcode. In some embodiments, an RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid pay load or a nucleic acid encoding pay load; and (c) a 3’ alphavirus UTR and an RNA barcode. In some embodiments, RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid pay load or a nucleic acid encoding pay load; and (c) a 3’ alphavirus UTR and an RNA barcode. In some embodiments, most of the RNA polynucleotides of a plurality of RNA polynucleotides comprise, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid pay load or a nucleic acid encoding pay load; and (c) a 3’ alphavirus UTR. In some embodiments, each of the RNA polynucleotides of a plurality of RNA polynucleotides

[0123] 12015157.1 comprise, from 5’ to 3’: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid pay load or a nucleic acid encoding pay load; and (c) a 3’ alphavirus UTR comprising an RNA barcode.

[0124] In some embodiments, this disclosure describes an RNA polynucleotide comprising: (a) a 5’ alphavirus UTR comprising a CSE; (b) a nucleic acid pay load or a nucleic acid encoding a payload; (c) an RNA barcode comprising at least 6 nucleotides; and (d) a 3’ alphavirus UTR.

[0125] In some embodiments, this disclosure describes an RNA polynucleotide comprising: (i) a 5’-SINV-UTR comprising a SINV CSE; (ii) a nucleic acid pay load or a nucleic acid encoding a pay load; (iii) an RNA barcode comprising at least 6 nucleotides; and (iv) a 3’-SINV-UTR.

[0126] In some embodiments, this disclosure describes an RNA polynucleotide comprising, from 3’ to 5’: (i) a 5’-SINV-UTR comprising a SINV CSE; (ii) a nucleic acid payload or a nucleic acid encoding a pay load; (iii) an RNA barcode comprising at least 6 RNA nucleotides; and (iv) a 3’-SINV-UTR.

[0127] In some embodiments, this disclosure describes an RNA polynucleotide comprising, from 3’ to 5’: (i) a 5’-SINV-UTR comprising a SINV CSE; (ii) a nucleic acid payload or a nucleic acid encoding a payload; (iii) a restriction site (e.g., a Bsal site for introducing an RNA barcode); and (iv) a 3’-SINV-UTR.

[0128] In some embodiments, this disclosure describes an RNA polynucleotide comprising, from 5’ to 3’: (i) a 5'-SINV-UTR comprising a SINV CSE; (ii) a payload;(iii) an RNA barcode comprising at least 6 nucleotides in the following sequence context CUCUACAAAUGAUAAUAG-RNA barcode-UCGAGGCGGCCGCCACGC (SEQ ID NO: 429); and (iv) a 3'-SINV-UTR. The skilled artisan will appreciate that the recitation of an “RNA barcode” in SEQ ID NO: 429 represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length. In some embodiments, SEQ ID NO: 429represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length and otherwise conforming to the definition of “RNA barcode” as used throughout the instant disclosure.

[0129] In some embodiments, this disclosure describes an RNA polynucleotide comprising, from 5’ to 3’: (i) a 5’-SINV-UTR comprising a SINV CSE; (ii) a nucleic acid payload or a nucleic acid encoding a pay load; and (iii) a 3’-SINV-UTR comprising an RNA barcode comprising at least 6 RNA nucleotides.

[0130] In some embodiments, this disclosure describes an RNA polynucleotide comprising, from 5’ to 3’: (i) a 5’-SINV-UTR comprising a SINV CSE; (ii) a nucleic acid payload or a nucleic acid encoding a pay load; and (iii) a 3’-SINV-UTR comprising an RNA barcode comprising at least 6 nucleotides, wherein the RNA barcode is present in the 3 ’-SINV -UTR in

[0131] 12015157.1 the following sequence context CUCUACAAAUGAUAAUAG-RNA barcode- UCGAGGCGGCCGCCACGC (SEQ ID NO: 429). The skilled artisan will appreciate that the recitation of an “RNA barcode” in SEQ ID NO: 429 represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length. In some embodiments, SEQ ID NO: 429represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length and otherwise conforming to the definition of “RNA barcode” as used throughout the instant disclosure.

[0132] In some embodiments, this disclosure describes an RNA polynucleotide comprising, from 5’ to 3’: (i) a 5’-SINV-UTR comprising a SINV CSE; (ii) a nucleic acid payload or a nucleic acid encoding a pay load; and (iii) a 3’-SINV-UTR comprising an RNA barcode comprising at least 6 nucleotides, the 3’-SINV-UTR comprising the sequence set forth in any one of SEQ ID NOs: 9-428.

[0133] In some embodiments, this disclosure describes an RNA polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. taRNA-based Screening of Delivery Vectors

[0134] In some aspects, RNA barcodes are useful for identifying (e.g., evaluating, characterizing) one or more qualities of a delivery vehicle (e.g., suitability of the delivery vehicle for delivering RNA cargo to the cytosol of a cell). A delivery vehicle may be any vehicle suitable for delivery of RNA to cells (e.g., cells of a subject).

[0135] Nanoparticle Screening

[0136] In some embodiments, a delivery vehicle is a nanoparticle. In some embodiments, RNA barcodes are useful in a method of identifying one or more qualities of a nanoparticle (e.g., suitability for delivering RNA cargo to the cytosol of a cell). In some embodiments, an RNA barcode in an RNA polynucleotide is associated with a nanoparticle comprising the RNA polynucleotide (e.g., bound to or encapsulating the RNA polynucleotides). In some embodiments, an RNA barcode in an RNA polynucleotide is indicative of the type of nanoparticle comprising (e.g., bound to or encapsulating) an RNA polynucleotide. In this way, detection of a given RNA barcode in cells of a specific cell type (e.g., amplification of the RNA barcode) may be used to determine which characteristics of nanoparticles (e.g., types, formulations) increase amplification of the RNA in a specific cell type.

[0137] A nanoparticle can be understood to be of a certain “type,” here referring to the type of components of which the nanoparticle is comprised (e.g., polymers, lipids). In some embodiments, a nanoparticle is a polymeric nanoparticle (i.e., composed primarily of polymers),

[0138] 12015157.1 a lipid nanoparticle (i.e., composed primarily of lipids), a hybrid nanoparticle (i.e., composed of a mixture of components, such as polymers and lipids), or a drug-conjugate nanoparticle (i.e., a nanoparticle conjugated to a drug).

[0139] In some embodiments, a nanoparticle is a polymeric nanoparticle. In some embodiments, nanoparticles are polymeric nanoparticles e.g., as described by Jiang X et al., Reference Module in Materials Science and Materials Engineering. 2021:B978-0-12-822425-0.00017-8.

[0140] In some embodiments, a nanoparticle is a lipid nanoparticle (LNP). In some embodiments, LNPs comprises an ionizable lipid, a phospholipid, cholesterol, and a polyethylene glycol (PEG)-ylated lipid (also known as a “PEG-lipid”). Lipid nanoparticles for RNA polynucleotide delivery are known in the art e.g., as described in Jung HN Theranostics. 2022 Oct 24;12(17):7509-7531; Paunovska, K., Nat Rev Genet 23, 265-280 (2022); and by Han, X., et al., Nat Commun 12, 7233 (2021). In some embodiments, an LNP comprises an ionizable lipid. Jung also describes LNP components, production, formulations, and RNA delivery. In some embodiments, LNPs of the plurality of LNPs comprise an ionizable lipid. In some embodiments, the ionizable lipid is an unsaturated ionizable lipid, a multi-tail ionizable lipid, a polymeric ionizable lipid, a biodegradable ionizable lipid and / or a branched-tail ionizable lipid. In some embodiments, the ionizable lipid is selected from Pig. 1 or Pig. 2 of Han, X., et al., Nat Commun 12, 7233 (2021). Pigs. 1 and 2 of Han, X., et al., Nat Commun 12, 7233 (2021) are incorporated by reference their entirety. In some embodiments, the unsaturated ionizable lipid comprises Dlin-MC3-DMA (i.e., MC3), OP-O2, A6, or A18-Iso2DC18. In some embodiments, the multi-tail ionizable lipid comprises 98N12-5, C12-200, CKK-E12, or 9A1P9. In some embodiments, the ionizable polymer- lipid comprises 7C1 or G0-C14. In some embodiments, the biodegradable ionizable lipid comprises L319, 304013, C12-200, OP-Deg- Lin, or 306-O12B. In some embodiments, the branched-tail ionizable lipid is 3060iio or PTT5. In some embodiments, the ionizable lipid comprises SM-102, ALC-0315, Acuitas A9, Lipid 2,2 (8,8) 4C CH3, Genevant CL1, LP000001, LP01, or MC3.

[0141] A nanoparticle can also be understood to have a certain “formulation,” here referring to the components of which the nanoparticle (absent of any RNA cargo for delivery) is comprised, in the relative amounts (e.g., ratios, molar ratios, percentages) in which the components are present. In some embodiments, a nanoparticle formulation comprises at least one component (e.g., at least one polymer, at least one lipid). In some embodiments, a nanoparticle formulation comprises two or more components. The skilled artisan will appreciate that nanoparticles consisting of the same components but differing in relative amounts of the components (e.g.,

[0142] 12015157.1 consisting of the same first component and the same second component, but differing in ratio of the first component:the second component) do not have the same formulation. As used herein, the term “component” may also be used to refer to ligands. In some embodiments, a nanoparticle comprises (e.g., is conjugated to) a ligand. In some embodiments, a ligand is a targeting moiety. Ligands and targeting moieties are known in the art, for example, as described by Yoo, J., et al. (2019). “Active targeting strategies using biological ligands for nanoparticle drug delivery systems.” Cancers, 11(5), 640.

[0143] The skilled artisan will appreciate that the type and / or formulation of a nanoparticle may be variably effective for releasing RNA cargo into the cytosol of a specific cell type. Different nanoparticles may target different specific cell types. For example, some nanoparticles generally target liver cells, whereas other nanoparticles target other organs, e.g., kidneys. In some aspects, RNA barcodes are useful for screening nanoparticles based on the cell types to which they are suitable for targeting. Without wishing to be bound to theory, nanoparticle formulations that increase RNA polynucleotide amplification may do so by increasing the amount of an RNA cargo, for example, RNA polynucleotides (e.g., trRNA, taRNA, or saRNA), that (1) enter the specific cell type; and (2) escapes the endosome and reaches the cytosol of the cell where amplification can occur.

[0144] In some aspects, provided herein is a method of determining delivery of an RNA polynucleotide by a nanoparticle to the cytosol of cells of a specific cell type and / or determining in vivo amplification of an RNA polynucleotide in cells of a specific cell type. In some embodiments, the method comprises obtaining a nanoparticle comprising an RNA polynucleotide comprising a CSE and an RNA barcode; contacting cells of a specific cell type with the nanoparticle, and determining delivery of the RNA polynucleotide to the cytosol of the cells of the specific cell type by detecting the RNA barcode in the cytosol of the cells. In some embodiments, the method comprises obtaining a nanoparticle comprising an RNA polynucleotide comprising a CSE and an RNA barcode; contacting cells of a specific cell type with the nanoparticle, and determining in vivo amplification of the RNA polynucleotide by detecting the RNA barcode in the cells of the specific cell type. In some embodiments, determining delivery of an RNA polynucleotide to the cytosol of a cell comprises determining (e.g., measuring) in vivo amplification of the RNA polynucleotide in the cell. Methods of determining delivery to the cytosol and in vivo amplification are described herein at least in the section entitled “Determining in vivo Amplification.”

[0145] 12015157.1 In some aspects, provided herein is a method of determining delivery of RNA polynucleotides of a plurality of RNA polynucleotides by a nanoparticle to the cytosol of cells of a specific cell type of a subject. In some embodiments, the method comprises: (i) obtaining a plurality of nanoparticles, each nanoparticle comprising an RNA polynucleotide of the plurality of the RNA polynucleotides, each RNA polynucleotide comprising a CSE and an RNA barcode; (ii) administering nanoparticles of the plurality of nanoparticles to the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining delivery of the RNA polynucleotides of the administered nanoparticles to the cytosol of the specific cell type by detecting the RNA barcode in the cytosol of the extracted cells of the specific cell type.

[0146] In some aspects, provided herein is a method of determining in vivo amplification of RNA polynucleotides of a plurality of RNA polynucleotides in cells of a specific cell type of a subject. In some embodiments, the method comprises: (i) obtaining a plurality of nanoparticles, each nanoparticle comprising an RNA polynucleotide of the plurality of the RNA polynucleotides, each RNA polynucleotide comprising a CSE and an RNA barcode; (ii) administering nanoparticles of the plurality of nanoparticles to the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining in vivo amplification of the RNA polynucleotides of the administered nanoparticles in the specific cell type by detecting the RNA barcode in the cytosol of the extracted cells of the specific cell type.

[0147] In some aspects, provided herein is a method of determining suitability of a nanoparticle formulation for delivery of RNA cargo the cytosol of cells of a specific cell type. In some embodiments, the method comprises: a step of obtaining a first plurality of nanoparticles and a second plurality of nanoparticles, wherein the first plurality of nanoparticles has a formulation comprising at least one different component relative to the second plurality of nanoparticles; wherein the first plurality of nanoparticles comprises a first RNA polynucleotide comprising a first RNA barcode and the second plurality of nanoparticles comprises a second RNA polynucleotide comprising a second RNA barcode; and wherein the first and second RNA barcodes are different; a step of contacting the cells of the specific cell type (e.g., administering to a subject in which the cells are comprised) the first and second pluralities of nanoparticles to the subject; and a step of determining that at least one of the first and / or second RNA polynucleotides was delivered to the cytosol of cells of the specific cell type by identifying the RNA barcode present in the cytosol of the cells of the specific cell type. In some embodiments

[0148] 12015157.1 this method may be performed using at least 3 (e.g., at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or at least 1000) different nanoparticles each having a formulation comprising at least one different component relative to the other three or more different nanoparticles, and each of the at least three or more different nanoparticles being associated with a different barcode. FIGs. 4-6 and 8 further illustrate exemplary methods by which nanoparticles may be screened for suitability to delivery cargo to certain cell types.

[0149] In some embodiments, the methods described herein comprise determining how well new and / or untested nanoparticles deliver RNA polynucleotides to the cytosol of cells and / or are amplified in vivo in the cells, and to which specific cell type(s) the RNA polynucleotides are delivered. In some embodiments, the method comprises determining suitability for delivery of RNA cargo to the cytosol (e.g., in vivo amplification of a given RNA polynucleotide) using different nanoparticle types. In some embodiments, the methods described herein are used to determine the suitability of at least 2 different nanoparticle types (e.g., polymer nanoparticles and lipid nanoparticles). In some embodiments, the methods described herein are used to determine the suitability of at least 2 different nanoparticle formulations (e.g., at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500 different nanoparticle types). In some embodiments, the different nanoparticle formulations comprise 2-400 different nanoparticle formulations. In some embodiments, the different nanoparticle formulations comprise 2-500 different nanoparticle formulations. In some embodiments, the different nanoparticle formulations comprise 10-400 different nanoparticle formulations. In some embodiments, the different nanoparticle formulations comprise 10-500 different nanoparticle formulations. In some embodiments, the different nanoparticle formulations comprise 50-400 different nanoparticle formulations. In some embodiments, the different nanoparticle formulations comprise 50-500 different nanoparticle formulations. In some embodiments, nanoparticles comprise 40-55% Ionizable Lipid (IL), 1.5-2.5% PEG-lipid , 8-15% phospholipid distearoylphosphatidylcholine (DSPC), and remaining % cholesterol. In some embodiments, nanoparticles comprise 50 / 1.5 / 10 / 38.5 or 47.5 / 2.5 / 10 / 40 IL / PEG-lipid / DSPC / Cholesterol.

[0150] Viral Vector Screening

[0151] In some embodiments, a delivery vehicle is a viral vector. In some embodiments, RNA barcodes are useful in a method for identifying one or more qualities of a viral vector (e.g., suitability for delivering RNA cargo to the cytosol of a cell). In some embodiments, an RNA barcode in an RNA polynucleotide is associated with a viral vector comprising the RNA

[0152] 12015157.1 polynucleotide (e.g., containing or encoding the RNA polynucleotides). In some embodiments, an RNA barcode in an RNA polynucleotide is indicative of the type of viral vector comprising (e.g., containing or encoding the RNA polynucleotides) an RNA polynucleotide. In this way, detection of a given RNA barcode in cells of a specific cell type (e.g., amplification of the RNA barcode) may be used to determine which types of viral vector increase amplification of the RNA in a specific cell type.

[0153] Viral vectors for RNA polynucleotide delivery include but are not limited to adeno- associate viral vectors, lentiviral vectors, and adenoviral vectors e.g., as described in Bulcha, J.T., Sig Transduct Target Ther 6, 53 (2021). In some embodiments, the viral vector is a picomavirus, anellovirus, togavirus, lyssavirus, or flavivirus viral vector. In some embodiments, a viral vector is an engineered viral vectors e.g., engineered picornavirus vectors, engineered anellovirus vectors, engineered togavirus vectors, engineered lyssavirus vectors, engineered flavivirus vectors, engineered adeno-associate viral vectors, engineered lentiviral vectors, or engineered adenoviral vectors.

[0154] The skilled artisan will appreciate that the certain features of viral vector may be variably effective for releasing RNA cargo into the cytosol of a specific cell type (i.e., direct the vector’s tropism). For example, adeno-associated viruses typically comprise capsid proteins that enable delivery of nucleic acid cargos to certain cell types (e.g., see: Zincarelli, C., et al. (2008). “Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.” Molecular therapy, 16(6), 1073-1080); while AAV4 serotype capsid proteins are associated with tropism for the lung and heart, the same serotype has lower transduction efficiency for the liver. Modifications of capsid proteins may affect the tropism and efficacy of viral vectors for certain uses (e.g., treatment of certain disease), for example, as described by Biining, H., et al. (2019). “Capsid modifications for targeting and improving the efficacy of AAV vectors.” Molecular therapy Methods & clinical development, 12, 248-265; accordingly, methods for screening their suitability are highly desirable. In some aspects, RNA barcodes are useful for screening viral vectors based on the cell types to which they are suitable for targeting.

[0155] In some aspects, provided herein is a method of determining delivery of an RNA polynucleotide by a viral vector to the cytosol of cells of a specific cell type and / or determining in vivo amplification of an RNA polynucleotide in cells of a specific cell type. In some embodiments, the method comprises obtaining a viral vector comprising an RNA polynucleotide comprising a conserved sequence element (CSE) and an RNA barcode; contacting cells of a specific cell type with the viral vector, and determining delivery of the RNA polynucleotide to

[0156] 12015157.1 the cytosol of the cells of the specific cell type by detecting the RNA barcode in the cytosol of the cells. In some embodiments, the method comprises obtaining a viral vector comprising an RNA polynucleotide comprising a conserved sequence element (CSE) and an RNA barcode; contacting cells of a specific cell type with the viral vector, and determining in vivo amplification of the RNA polynucleotide by detecting the RNA barcode in the cells of the specific cell type. Methods of determining delivery to the cytosol and in vivo amplification are described herein at least in the section entitled “Determining in vivo Amplification.”

[0157] In some aspects, provided herein is a method of determining delivery of a plurality of RNA polynucleotides by a viral vector to the cytosol of cells of a specific cell type of a subject. In some embodiments, the method comprises: (i) obtaining a plurality of viral vectors, each viral vector comprising an RNA polynucleotide of the plurality of the RNA polynucleotides, each RNA polynucleotide comprising a CSE and an RNA barcode; (ii) administering viral vectors of the plurality of viral vectors to the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining delivery of the RNA polynucleotides of the administered viral vectors to the cytosol of the specific cell type by detecting the RNA barcode in the cytosol of the extracted cells of the specific cell type.

[0158] In some aspects, provided herein is a method of determining in vivo amplification of RNA polynucleotides of a plurality of RNA polynucleotides in cells of a specific cell type of a subject. In some embodiments, the method comprises: (i) obtaining a plurality of viral vectors, each viral vector comprising an RNA polynucleotide of the plurality of the RNA polynucleotides, each RNA polynucleotide comprising a CSE and an RNA barcode; (ii) administering viral vectors of the plurality of viral vectors to the subject; (iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and (iv) determining in vivo amplification of the RNA polynucleotides of the administered viral vectors to the cytosol of the specific cell type by detecting the RNA barcode in the cytosol of the extracted cells of the specific cell type.

[0159] In some aspects, provided herein is a method of determining suitability of a viral vector formulation for delivery of RNA cargo the cytosol of cells of a specific cell type. In some embodiments, the method comprises: a step of obtaining a first plurality of viral vectors and a second plurality of viral vectors, wherein the first plurality of viral vectors has a formulation comprising at least one different component relative to the second plurality of viral vectors; wherein the first plurality of viral vectors comprises a first RNA polynucleotide comprising a

[0160] 12015157.1 first RNA barcode and the second plurality of viral vectors comprises a second RNA polynucleotide comprising a second RNA barcode; and wherein the first and second RNA barcodes are different; a step of contacting the cells of the specific cell type (e.g., administering to a subject in which the cells are comprised) the first and second pluralities of viral vectors to the subject; and a step of determining that at least one of the first and / or second RNA polynucleotides was delivered to the cytosol of cells of the specific cell type by identifying the RNA barcode present in the cytosol of the cells of the specific cell type. In some embodiments this method may be performed using at least 3 (e.g., at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or at least 1000) different viral vectors each being derived from different viruses or comprising at least one modification (e.g., different capsid protein) relative to the other three or more different viral vectors, and each of the at least three or more different viral vectors being associated with a different barcode.

[0161] In some embodiments, the methods described herein comprise determining how well new and / or untested viral vectors deliver RNA polynucleotides to the cytosol of cells, and to which specific cell type(s) the RNA polynucleotides are delivered. In some embodiments, the method comprises determining suitability for delivery of RNA cargo to the cytosol (e.g., in vivo amplification of a given RNA polynucleotide) using different viral vectors. In some embodiments, the methods described herein are used to determine the suitability of at least 2 different viral vectors (e.g., AAV vectors having different capsid protein modifications).

[0162] Obtaining a Plurality of RNA polynucleotides

[0163] In some embodiments, a method of determining in vivo amplification of a plurality of RNA polynucleotides in a specific cell type of a subject comprises obtaining a plurality of RNA polynucleotides.

[0164] A “plurality” refers to at least 2. In some embodiments, a plurality comprises at least 5 (e.g., at least 10, at least 100, at least 1,000, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10).

[0165] A “plurality of RNA polynucleotides” refers to at least 2 RNA polynucleotides (e.g., at least 2 trans-replicating RNAs). In some embodiments, a plurality of RNA polynucleotides comprises at least 5 (e.g., at least 10, at least 100, at least 1,000, at least IxlO4, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, or at least IxlO10) RNA polynucleotides. In some embodiments, the plurality of RNA polynucleotides comprises at least 2 RNA polynucleotides with different sequences (e.g., at least 5 polynucleotides with different sequences, at least 10 polynucleotides with different sequences, at least 25 polynucleotides with

[0166] 12015157.1 different sequences, at least 50 polynucleotides with different sequences, at least 75 polynucleotides with different sequences, at least 100 polynucleotides with different sequences, at least 125 polynucleotides with different sequences, at least 150 polynucleotides with different sequences, at least 200 polynucleotides with different sequences, at least 300 polynucleotides with different sequences, at least 400 polynucleotides with different sequences, at least 500 polynucleotides with different sequences, at least 750 polynucleotides with different sequences, at least 5 polynucleotides with different sequences, or at least 1000 polynucleotides with different sequences). In some embodiments, the plurality of RNA polynucleotides comprises 2- 500 RNA polynucleotides with different sequences. In some embodiments, the plurality of RNA polynucleotides comprises 2-400 RNA polynucleotides with different sequences. In some embodiments, the plurality of RNA polynucleotides comprises 10-500 RNA polynucleotides with different sequences. In some embodiments, the plurality of RNA polynucleotides comprises 10-400 RNA polynucleotides with different sequences. In some embodiments, the plurality of RNA polynucleotides comprises 50-500 RNA polynucleotides with different sequences. In some embodiments, the plurality of RNA polynucleotides comprises 50-400 RNA polynucleotides with different sequences. In some embodiments, 2 or more RNA polynucleotides having different sequences each comprise a different RNA barcode. In some embodiments, 2 or more RNA polynucleotides having different sequences have different RNA barcodes but otherwise have identical sequences (e.g., identical 5’ UTRs, identical CSEs, identical nucleic acids encoding a pay load).

[0167] A plurality of RNA polynucleotides may be obtained in any suitable way. In some embodiments, obtaining the plurality of RNA polynucleotides comprises synthesizing the plurality of RNA polynucleotides. In some embodiments, obtaining the plurality of RNA polynucleotides comprises synthesizing a plurality of DNA sequences that when transcribed produces the plurality of RNA polynucleotides. Methods of DNA polynucleotide synthesis and RNA polynucleotide synthesis are known in the art and include, but are not limited to, silicon- based synthesis or phosphonamidite synthesis (e.g., as described in Hoose, A. Nat Rev Chem 7, 144-161 (2023)). Depending on the length of the polynucleotide, the method may comprise synthesizing portions of a given polynucleotide then ligating those portions together (e.g., as described in Hoose, A. Nat Rev Chem 7, 144-161 (2023)). In some embodiments, obtaining the plurality of RNA polynucleotides comprises determining the sequences of the plurality of RNA polynucleotides and obtaining synthesized polynucleotides comprising the sequences (e.g., instructing a third party to synthesize the sequences). In some embodiments, obtaining the

[0168] 12015157.1 plurality of RNA polynucleotide comprises obtaining a plurality of plasmids that comprise DNA, that when transcribed, produces the plurality of RNA polynucleotides. In some embodiments, obtaining the plurality of RNA polynucleotide comprises obtaining a plurality of vectors (e.g., viral vectors) that comprise DNA, that when transcribed, produces the plurality of RNA polynucleotides. In some embodiments, obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of vectors (e.g., viral vectors) that comprises the RNA polynucleotides of the plurality of RNA polynucleotides. In some embodiments, obtaining the plurality of RNA polynucleotides comprising obtaining a plurality of DNA polynucleotides (e.g., plasmids) that when transcribed produce the plurality of RNA polynucleotides, and performing in vitro transcription on the plurality of plasmids. The skilled artisan will appreciate that the RNA sequences provided herein (e.g., in Tables 1 and 2) also encompass DNA sequences used to generate RNA polynucleotides having the RNA sequences (e.g., for in vitro transcription); for example, any one or more of the uracil bases (U’s) in any one of the RNA polynucleotide sequences provided herein may optionally be a thymine bases (T’s).

[0169] In some embodiments, obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of RNA polynucleotides and obtaining additional polynucleotides that are not part of the plurality of polynucleotides (e.g., polynucleotides that do not comprise an RNA barcode and / or a CSE that is cognate to the replicase).

[0170] In some embodiments, obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of nanoparticles, wherein nanoparticles of the plurality of nanoparticles comprise an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, most nanoparticles of the plurality of nanoparticles comprises an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, each nanoparticle of the plurality of nanoparticles comprises an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, nanoparticles of the plurality of nanoparticles comprise a replicase construct encoding a replicase that is cognate to the CSE of the RNA polynucleotides.

[0171] In some embodiments, obtaining a plurality of nanoparticles comprises obtaining nanoparticles comprising taRNAs (e.g., an RNA polynucleotide comprising a CSE and RNA barcode and a separate RNA polyribonucleotide comprising a nucleic acid encoding a replicase). In some embodiments, obtaining a plurality of nanoparticles comprises obtaining nanoparticles comprising saRNAs (e.g., an RNA polynucleotide comprising a CSE, an RNA barcode, and a nucleic acid encoding a replicase). In some embodiments, the method further comprises

[0172] 12015157.1 obtaining a first plurality of nanoparticles that comprises an RNA polynucleotide comprising a nucleic acid encoding a replicase and not comprising an RNA polynucleotide comprising a CSE and an RNA barcode.

[0173] In some embodiments, obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of viral vectors, wherein viral vectors of the plurality of viral vectors comprise or encode (i.e., comprise DNA polynucleotides encoding) an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, most viral vectors of the plurality of viral vectors comprises an RNA polynucleotide of the plurality of RNA viral vectors. In some embodiments, each viral vector of the plurality of viral vectors comprises or encodes (i.e., comprises DNA polynucleotides encoding) an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, viral vectors of the plurality of viral vectors comprise or encode (i.e., comprise DNA polynucleotides encoding) a replicase construct encoding a replicase that is cognate to the CSE of the RNA polynucleotides.

[0174] Contacting In Vivo

[0175] In some embodiments, the method of determining in vivo amplification of a plurality of RNA polynucleotides in a specific cell type of a subject comprises (i) obtaining the plurality of RNA polynucleotides, as described herein; and (ii) contacting, in vivo, RNA polynucleotides of the plurality of RNA polynucleotides with cells of the specific cell type of the subject.

[0176] In some embodiments, contacting comprises physical interaction between an RNA polynucleotide (e.g., of a plurality of RNA polynucleotides) and a cell of the specific cell type. In some embodiments, the physical interaction is transitory. In some embodiments, the physical interaction results in the RNA polynucleotide entering the cell. In some embodiments, when an RNA polynucleotide (e.g., a plurality of RNA polynucleotides) contacts a cell (e.g., cells) of the specific cell type, some physical interactions between the RNA polynucleotides and the cell are transitory and other physical interactions result in the RNA polynucleotide entering the cell.

[0177] In some embodiments, contacting comprises contacting a plurality of delivery vehicles (e.g., nanoparticles or viral vectors) that comprise RNA polynucleotides of a plurality of RNA polynucleotides with cells of a specific cell type. In some embodiments, a delivery vehicle of the plurality of delivery vehicles comprises an RNA polynucleotide of the plurality of RNA polynucleotides. In some embodiments, delivery vehicles of the plurality of delivery vehicles comprise RNA polynucleotides of the plurality of RNA polynucleotides. In some embodiments, most of the delivery vehicles of a plurality of delivery vehicles comprise RNA polynucleotides of the plurality of RNA polynucleotides. In some embodiments, all of the delivery vehicles

[0178] 12015157.1 comprise one type of RNA polynucleotide. For example, a delivery vehicle may comprise 1000 RNA polynucleotides, wherein all the RNA polynucleotides have the same sequence.

[0179] In some embodiments, contacting comprises contacting (i) a delivery vehicle comprising an RNA polynucleotide of the plurality of RNA polynucleotides and (2) a cell of the specific cell type. In some embodiments, contacting comprises contacting (i) a nanoparticle comprising an RNA polynucleotide of the plurality of RNA polynucleotides and (2) a cell of the specific cell type. In some embodiments, contacting comprises contacting (i) a polymeric nanoparticle comprising an RNA polynucleotide of the plurality of RNA polynucleotides and (2) a cell of the specific cell type. In some embodiments, contacting comprises contacting (i) a lipid nanoparticle (LNP) comprising an RNA polynucleotide of the plurality of RNA polynucleotides and (2) a cell of the specific cell type. In some embodiments, contacting comprises contacting (i) a viral vector comprising an RNA polynucleotide of the plurality of RNA and (2) a cell of the specific cell type.

[0180] In some embodiments, contacting comprises administering a plurality of RNA polynucleotides (e.g., a plurality of delivery vehicles comprising the plurality of RNA polynucleotides) to a subject (e.g., a subject containing cells of the specific cell type). Administration may be performed by any suitable means. In some embodiments, the administration comprises oral administration. In some embodiments, the administration comprises intravenous administration. In some embodiments, the administration comprises intrathecal or intracranial administration. In some embodiments, administration comprises intramuscular injection. In some embodiments, administration comprises inhalation (e.g., intranasal administration). In some embodiments, administering RNA polynucleotides comprises administering delivery vehicles comprising the RNA polynucleotides. In some embodiments, administering RNA polynucleotides comprises administering nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles) comprising the RNA polynucleotides. In some embodiments, administering RNA polynucleotides comprises administering viral vectors comprising the RNA polynucleotides.

[0181] A “specific cell type” refers to a set of cells from a subject that have similar characteristics compared to a different set of cells from the same subject. The skilled artisan will appreciate that it may be desirable to target or exclude cells at different levels of specificity; for example, it may be desirable for a delivery vehicle to target cells of a specific organ, tissue, or subtype of cell for delivery of an RNA therapeutic. In some embodiments, the specific cell type is a cell type of a specific organ (e.g., brain, eyes, skin, spinal cord, peripheral nervous

[0182] 12015157.1 system, lung, heart, spleen, kidney, liver, intestine, testis, and ovaries). For example, liver cells may be considered a specific cell type (e.g., compared to kidney cells). In some embodiments, a specific cell type is cell type of a bodily fluid (e.g., bone marrow cells or blood cells). In some embodiments, a specific cell type comprises cells of a tissue of an organ. For example, stromal cells may be considered a specific cell type (e.g., compared to other liver cells). In some embodiments, the specific cell type comprises a subtype of cells in a specific tissue. For example, liver cells can be subdivided into different subtypes (e.g., hepatocytes, hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells).

[0183] A “subject” refers to a multi-cellular organism. In some embodiments, the subject is a mammal. In some embodiments, the subject is a rodent (e.g., a mouse or rat). In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a nonhuman animal. In some embodiments, the subject is a human. In some embodiments, the subject is an insect (e.g., Drosophila). In some embodiments, the subject is a plant (e.g., Arabidopsis).

[0184] In some embodiments, the cells of the specific cell type comprise a replicase that is cognate to CSE comprised in an RNA polynucleotide described herein (e.g., comprised in a delivery vehicle). In some embodiments, the cells have been genetically engineered to comprise DNA encoding a replicase construct (e.g., encoding an RNA polynucleotide encoding the replicase). In some embodiments, the cells comprise an RNA (e.g., an mRNA) encoding the replicase.

[0185] In some embodiments, a method provided herein comprises contacting cells of a specific cell type with an saRNA (i.e., an RNA polynucleotide comprising a CSE, an RNA barcode, and a nucleic acid encoding a replicase). In some embodiments, the method comprises contacting cells of the specific cell type with a plurality of saRNAs that each comprise an RNA polynucleotide of a plurality of RNA polynucleotides, each RNA polynucleotide comprising a CSE, an RNA barcode, and a nucleic acid encoding a replicase. In some embodiments, the method comprises contacting cells of a specific cell type with a plurality of delivery vehicles (e.g., lipid nanoparticles) that collectively comprise a plurality of saRNAs. In some embodiments, the method comprises administering to a subject a plurality of delivery vehicles (e.g., lipid nanoparticles) that collectively comprise a plurality of saRNAs. In some embodiments, most of the delivery vehicles of a plurality of delivery vehicles comprise one type of saRNA. For example, a given delivery vehicle may comprise 1000 saRNAs, wherein all the saRNAs have the same sequence.

[0186] 12015157.1 In some embodiments, a method provided herein comprises contacting cells of a specific cell type with a taRNA. In some embodiments, the method comprises contacting cells of a specific cell type with a plurality of taRNAs. In some embodiments, the method comprises contacting cells of a specific cell type with a plurality of delivery vehicles (e.g., lipid- nanoparticles) that collectively comprise a plurality of taRNAs. In some embodiments, the method comprises administering to a subject a plurality of delivery vehicles (e.g., lipid- nanoparticles) that collectively comprise a plurality of taRNAs. In some embodiments, most of the delivery vehicles of the plurality of delivery vehicles comprise one or fewer types of taRNAs.

[0187] In some embodiments, the method comprises contacting cells of the specific cell type with: (i) a first delivery vehicle comprising an RNA polynucleotide comprising a CSE and an RNA barcode and not comprising a replicase construct (e.g., a trRNA); and (ii) a second delivery vehicle comprising a replicase construct cognate to the CSE, but not comprising the RNA polynucleotide. In some embodiments, the method comprises contacting cells of the specific cell type with: (i) a first plurality of delivery vehicles collectively comprising a plurality of RNA polynucleotides comprising CSEs and RNA barcodes and not comprising replicase constructs; and (ii) a second plurality of delivery vehicles comprising a plurality of replicase construct, but not comprising an RNA polynucleotide. In some embodiments, the method comprises administering to a subject: (i) a first plurality of delivery vehicles collectively comprising a plurality of RNA polynucleotides comprising CSEs and RNA barcodes and not comprising a replicase construct; and (ii) a second plurality of delivery vehicles comprising a plurality of replicase constructs, but not comprising RNA polynucleotides comprising CSEs and RNA barcodes.

[0188] In some embodiments, the method comprises contacting cells of a specific cell type with: (i) a plurality of first nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles) collectively comprising a plurality of RNA polynucleotides comprising CSEs and RNA barcodes and not comprising the replicase construct; and (ii) a second plurality of nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles) comprising a plurality of replicase constructs, but not comprising RNA polynucleotides comprising CSEs and RNA barcodes. Nanoparticles comprising a polyribonucleotide encoding a replicase, but not comprising an RNA polynucleotide comprising a CSE and RNA barcode, may be referred to herein as a “replicase nanoparticle.” In some embodiments, the method comprises administering to a subject: (i) a plurality of first nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles) collectively

[0189] 12015157.1 comprising a plurality of RNA polynucleotides comprising CSEs and RNA barcodes and not comprising the replicase construct; and (ii) a second plurality of nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles) comprising a plurality of replicase constructs, but not comprising RNA polynucleotides comprising CSEs and RNA barcodes.

[0190] In some embodiments, the method comprises administering to a subject: (i) a plurality of first nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles) collectively comprising a plurality of RNA polynucleotides comprising CSEs and RNA barcodes and not comprising the replicase construct; and (ii) a second plurality of nanoparticles (e.g., polymer nanoparticles, lipid nanoparticles) comprising a plurality of replicase constructs, but not comprising RNA polynucleotides comprising CSEs and RNA barcodes.

[0191] In some embodiments, the method comprises administering to a subject: (i) a plurality of first viral vectors collectively comprising a plurality of RNA polynucleotides comprising CSEs and RNA barcodes and not comprising the replicase construct; and (ii) a second plurality of viral vectors comprising a plurality of replicase constructs, but not comprising RNA polynucleotides comprising CSEs and RNA barcodes.

[0192] In some embodiments, most of the delivery vehicles of the first plurality of delivery vehicles comprise one type of RNA polynucleotides (e.g., comprise RNA polynucleotides having the same sequences).

[0193] In some embodiments, a method provided herein comprises contacting cells of a specific cell type with an RNA polynucleotide comprising a CSE and RNA barcode (e.g., a trRNA), wherein cells of the specific cell type are engineered to express a cognate replicase (e.g., by genomic integration of a polynucleotide encoding the replicase). Methods of engineering multicellular organisms to express genes are known in the art, e.g., as described Fujii W et al., Methods Mol Biol. 2017;1630:91-100; Bosch JA Genetics. 2020 Jan;214(l):75-89; and Dong OX. Mol Plant. 2020 Aug 3; 13(8): 1104. For example, a specific cell type of a subject (e.g., a non-human subject) is recombinantly modified to express a replicase. In some embodiments, the subject is a mouse or rat and a specific cell type of the mouse or rat has been engineered to express a replicase.

[0194] In some embodiments, a method provided herein comprises contacting cells of a specific cell type with a plurality of RNA polynucleotides comprising a CSE and RNA barcode (e.g., a plurality of trRNAs), wherein cells of the specific cell type are engineered to express a cognate replicase. In some embodiments, the method comprises contacting cells of the specific cell type with (i) a first delivery vehicle comprising the RNA polynucleotide comprising a CSE and RNA

[0195] 12015157.1 barcode (e.g., a trRNA) and not comprising the replicase construct, wherein cells of the specific cell type are engineered to express a cognate replicase.

[0196] In some embodiments, an RNA polynucleotide comprises a nucleic acid encoding a payload, wherein the payload is a genome editing enzyme, and the method further comprising contacting cells of the plurality of cells with a polynucleotide comprising a nucleic acid that, when transcribed, produce a guide RNA, i.e., an RNA polynucleotide that can direct the genome editing agent to its target gene or transcript. In some embodiments, the RNA polynucleotide comprises the polynucleotide comprising a nucleic acid that, when transcribed, produces a guide RNA. In some embodiments, the delivery vehicle comprises the guide RNA. In some embodiments, the guide RNA is a single guide RNA. In some embodiments, the guide RNA comprises two different RNAs: a tracer RNA and a crRNA.

[0197] Extracting from the Subject

[0198] In some embodiments, a method provided herein comprises extracting, from a subject, cells of a specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to a CSE comprised in RNA polynucleotides utilized in the methods described herein.

[0199] “Extracting” refers to removing cells of the specific cell type from the subject. Extracting includes, but is not limited to, removing a biological sample from the subject that is expected to comprise the specific cell type. A biological sample refers to any portion of the subject. In some embodiments, the biological sample is an organ or a portion of an organ. In some embodiments, the biological sample is a bodily fluid or a portion of a bodily fluid. In some embodiments, extracting comprises biopsy of an organ or bodily fluid. In some embodiments, the biological sample is a sample of the subject’s brain, eyes, skin, spinal cord, peripheral nervous system, lung, heart, spleen, kidney, liver, intestine, testis, ovaries, blood, and / or bone marrow. In some embodiments, extracting comprises further isolating cells of the specific cell type from the biological sample. Isolating may be performed by any suitable means, including but not limited to, fluorescent assisted or magnetic cell sorting. In some embodiments, isolating is not a perfect isolation (i.e., does not result in 100% cells of the desired specific cell type). For example, isolating may comprise enriching for a particular cell type from a population of different cell types. In some embodiments, after isolation, the isolated cells are at least 90% the specific cell type (e.g., at least 95%, at least 98% or at least 99% the specific cell type).

[0200] As described above, cells of the specific cell type may comprise a replicase that is cognate to a CSE comprised in an RNA polynucleotide used in a method provided herein. In some embodiments, the replicase is expressed in cells from a replicase construct (e.g., as part of

[0201] 12015157.1 a saRNA or a taRNA). In some embodiments, the cells of the specific cell type are engineered to express the replicase. In some embodiments, the cells of the specific cell type are engineered to express the replicase construct prior to the contacting step.

[0202] Determining in vivo Amplification

[0203] In some embodiments, a method provided herein comprises determining in vivo amplification of a plurality of RNA polynucleotides comprising an RNA barcode in a subject by measuring an amount of each RNA barcode in extracted cells of the specific cell type.

[0204] Determining in vivo amplification of the plurality of RNA polynucleotides may be performed using any suitable means. In some embodiments, determining comprises detecting an RNA barcode in cells of the specific cell type. An RNA barcode is “detected” in a cell (e.g., in cells of a specific cell type” when it is measured at an amount indicative of the RNA barcode being present in the cell. In some embodiments, the amount is an amount above a baseline (e.g., a baseline of zero). In some embodiments, the amount is a statistically significant amount. An RNA barcode can be detected using any mean known in the art, for example, using nextgeneration sequencing, in situ hybridization (e.g., hybridization of the RNA barcodes to probes comprising a detectable moiety), targeted sequencing (e.g., Sanger sequencing), polymerase chain reaction (e.g., with a primer complementary to the RNA barcode)

[0205] In some embodiments, determining comprises measuring an amount of two or more RNA barcodes in the extracted cells of the specific cell type. In some embodiments, determining comprises measuring an amount of each RNA barcode in the extracted cells of the specific cell type. In some embodiments, determining comprises determining enrichment of one or more RNA barcodes in the extracted cells of the specific cell type (e.g., enrichment of one or more RNA barcodes of a plurality of RNA barcodes). In some embodiments, determining in vivo amplification comprises determining a difference in an amount of an RNA barcode in a cell of the specific cell type relative to an amount of the same RNA barcode in a control cell (e.g., a negative control cell, a positive control cell). In some embodiments, determining in vivo amplification comprises determining a difference in an amount of an RNA barcode in a cell of the specific cell type after contacting the cell with an RNA polynucleotide comprising the RNA barcode relative to an amount of the same RNA barcode in the cell before the contacting.

[0206] Measuring may be performed using any suitable technique for measuring the amount of each RNA barcode of the plurality of RNA barcodes. An “amount” of each RNA barcode refers to, for example the actual amount of the RNA barcode (e.g., in mols or grams), a count of RNA barcode copies, or a relative amount of each RNA barcode (e.g., as compared to a control, or

[0207] 12015157.1 total amount of RNA sequenced). A control may be, for example, a known amount of an RNA polynucleotide standard (e.g., an amount of the RNA barcode in a control cell, an amount of the RNA barcode expressed by the same cells before the contacting). In some embodiments, measuring comprises performing qPCR to determine the amount of an RNA barcode of an RNA polynucleotide. In some embodiments, measuring comprises performing qPCR to determine an amount of most of the RNA barcodes of a plurality of RNA polynucleotides. In some embodiments, measuring comprises performing qPCR to determine amounts of each RNA barcode of a plurality of RNA polynucleotides.

[0208] In some embodiments, measuring comprises performing next-generation sequencing (NGS) (e.g., ILLUMINA sequencing) to determine an amount of an RNA barcode of an RNA polynucleotide. In some embodiments, measuring comprises performing NGS to determine an amount of most of the RNA barcodes of the plurality of RNA polynucleotides. In some embodiments, measuring comprises performing NGS to determine an amount of each of the RNA barcodes of the plurality of RNA polynucleotides. In some embodiments, the method comprising preparing the RNA polynucleotides extracted from the cells of the specific cell type for NGS.

[0209] Preparing RNA polynucleotides for NGS may be done using any suitable method. Methods of preparing RNA polynucleotides for NGS are known in the art. In some embodiments, preparing RNA polynucleotides comprises reverse transcribing an RNA barcode into deoxyribonucleic acid (DNA) polynucleotides. In some embodiments, the method comprises ligating a unique molecule identifier (UMI) to extracted RNA polynucleotides of a plurality of RNA polynucleotide or to DNA reverse transcribed from RNA polynucleotides of a plurality of RNA polynucleotides. In some embodiments, the method comprising amplifying a DNA polynucleotide (e.g., reverse transcribed from the RNA polynucleotide) comprising the UMI.

[0210] Performing NGS to measure the amount polynucleotides of the plurality of RNA polynucleotides produces sequencing data. In some embodiments, the sequencing data comprises counts of each instance an RNA barcode is observed in the sample that was sequenced. In some embodiments, the sequencing data comprises counts of instances a control sequence or RNA barcode is observed in the sequencing data. In some embodiments, the sequencing data comprises a count of the total number of polynucleotides sequenced. In some embodiments, analyzing the sequencing data comprises normalizing a count of instances a given RNA barcode is observed in a sample by a count of instances a control RNA sequence is

[0211] 12015157.1 observed to determine a relative amount of the given RNA barcode. In some embodiments, analyzing the sequencing data comprises normalizing the count of instances a given RNA barcode is observed by a count of the total number of RNA polynucleotides (reversed transcribed to DNA) sequenced to determine a relative amount of the given RNA barcode.

[0212] In some embodiments, a method provided herein comprises sequencing a plurality of RNA polynucleotides before a contacting step and after an extracting step (e.g., before and after administration of the plurality of RNA polynucleotides to the subject), and then determining a change in the relative amount of at least some of the RNA barcodes (e.g., one or more, two or more of, most of, or all the RNA barcodes) after the extracting step compared to before the contacting step. In some embodiments, an increase in the relative amount of a given RNA barcode indicates that the RNA polynucleotide comprising the RNA barcode has been amplified. In some embodiments, no change in the relative amount of a given RNA barcode indicates that the RNA polynucleotide comprising the RNA barcode has not been amplified. In some embodiments, a decrease in the relative amount of a given RNA barcode indicates that the RNA polynucleotide comprising the RNA barcode has degraded or not been delivered to cells of the specific cell type.

[0213] In some embodiments, the method comprises determining the effects of different experimental variables (e.g., different delivery vehicle types or different RNA polynucleotide types ) on RNA barcode amplification. For example, a plurality of RNA polynucleotides may comprise many copies of the same RNA polynucleotide that contact cells of a specific cell type using a plurality of chemically different nanoparticles (i.e., nanoparticles having different formulations), e.g., 2-400 chemically different nanoparticles. In this example, a difference in amplification of different RNA polynucleotides can be associated with the different chemical characteristics of the nanoparticles (e.g., differences in components, differences in relative amounts of components). In some embodiments, this method comprises identifying lipid nanoparticle chemistries (i.e., formulations) that increase amplification of RNA polynucleotide (e.g., increase delivery of RNA cargoes to a cell of a specific cell type). The plurality of chemically different nanoparticles may comprise a plurality of chemically different types of nanoparticles (e.g., polymeric nanoparticles and lipid nanoparticles). The plurality of chemically different nanoparticles may comprise a plurality of chemically different polymeric nanoparticles. The plurality of chemically different nanoparticles may comprise a plurality of chemically different lipid nanoparticles. In another example, the plurality of polynucleotides comprises many copies of the same polynucleotide that are contacted to cells of the specific cell

[0214] 12015157.1 type using a plurality of different viral vectors (e.g., 2-400 chemically different viral vectors). In some embodiments, the viral vector comprises an adeno-associated viral vector, a lentiviral vector, an adenovirus, or a modified variant thereof. As with nanoparticles, in some embodiments, an increase in RNA polynucleotide amplification identifies a corresponding viral vector or modifications thereof (e.g., capsid protein modifications) that increases delivery of the RNA polynucleotide to the cytosol of the cells of the specific cell type.

[0215] In some embodiments, the method comprising contacting a plurality of RNA polynucleotides comprising RNA polynucleotides with different mutations with cells of the specific cell type using a plurality of delivery vehicles of the same type (e.g., a plurality of nanoparticles having the same or very similar chemical composition). For example, RNA polynucleotides of the plurality of polynucleotides may comprise mutations in the alphavirus 3’- UTR, alphavirus 5’-UTR, the pay load, and / or the CSE. These mutations may be any type of mutation, including but not limited to, point mutations, insertions, deletions, inversions, and trans versions. In some embodiments, the mutations are made randomly. In some embodiments, the mutations are made based on predicted effects (e.g., a prediction that a mutation at a particle position will increase in vivo amplification). In some embodiments, the mutations are directed to a particular portion of the RNA polynucleotide (e.g., in a stem loop of a UTR).

[0216] Without wishing to be bound to theory, a change in amplification of a particular RNA polynucleotide may be due toa mutation made to that particular RNA polynucleotide. In some embodiments, the method further comprises identifying RNA polynucleotide mutations that alter RNA polynucleotide amplification in vivo in the specific cell type. In some embodiments, the method further comprises identifying RNA polynucleotide mutations that increase RNA polynucleotide amplification in vivo in the specific cell type. In some embodiments, the method further comprises identifying RNA polynucleotide mutations that decrease RNA polynucleotide amplification in vivo in the specific cell type.

[0217] Preparing Compositions for Delivery of RNA Cargo

[0218] In some aspects, provided herein is a method of preparing compositions for delivery of RNA cargo to the cytosol of cells of a specific cell type in a subject. As used herein, the term “RNA cargo” refers to any RNA to be delivered to a cell, for example, therapeutic RNA (e.g., RNAs encoding a therapeutic payload) and functional RNA. RNA cargo may be comprised in any delivery vehicle (e.g., nanoparticle, viral vectors).

[0219] In some embodiments, the method comprises: a step of obtaining a first plurality of delivery vehicles and a second plurality of delivery vehicles, wherein the first plurality of

[0220] 12015157.1 delivery vehicles is different relative to the second plurality of delivery vehicles; wherein the first plurality of delivery vehicles comprises a first RNA polynucleotide comprising a first RNA barcode and the second plurality of delivery vehicles comprises a second RNA polynucleotide comprising a second RNA barcode, and wherein the first and second RNA barcodes are different; a step of administering the first and second pluralities of nanoparticles to a subject; a step of determining that at least one of the first and / or second RNA polynucleotides was delivered to the cytosol of cells of the specific cell type by identifying the RNA barcode present in the cytosol of the cells of the specific cell type; and a step of preparing a composition comprising a plurality of delivery vehicles comprising RNA cargo and having the formulation of the nanoparticle comprising the RNA barcode identified as delivered to the cytosol of the cells of the specific cell type. In some embodiments, the delivery vehicles are nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles). In some embodiments, the delivery vehicles are viral vectors. Selected delivery vehicles may be combined, for example, with pharmaceutically acceptable carrier.

[0221] EXEMPLARY SEQUENCES

[0222] Table 1: Exemplary trRNA and UTR Sequences

[0223] 12015157.1

[0224] 12015157.1

[0225] 12015157.1

[0226] 12015157.1

[0227] 12015157.1

[0228] The skilled artisan will appreciate that the recitations of an “RNA barcode” in SEQ ID NOs: 7-8 represent any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length and otherwise conforming to the definition of “RNA barcode” as used throughout the instant disclosure. Table 2: Barcode DNA sequences

[0229] 12015157.1

[0230] 12015157.1

[0231] 12015157.1

[0232] 12015157.1

[0233] 12015157.1

[0234] 12015157.1

[0235] 12015157.1

[0236] 12015157.1 EXAMPLES

[0237] Example 1. Design of Barcoded taRNA for Nanoparticle Screening

[0238] For nucleic acid therapeutics (e.g. mRNA, siRNA, ASOs, expressing DNA) to function, they must arrive at the cell-type of interest (biodistribution) and escape the endosomal uptake to be introduced into the cytosol (endosomal escape / cytosolic delivery). These are the two primary barriers to in vivo “functional delivery” - biodistribution and delivery.

[0239] Previous methods have been developed to test multiple (10s - 100s) nanoparticles in the same animal through various forms of nucleic acid barcoding. However, these existing methods only measure biodistribution and do not comment on endosomal escape efficiency. For example, some screening systems (e.g. Sago PNAS 2018) co-deliver various reporter RNAs (e.g., Cre, aVHH) with an oligonucleotide DNA barcode; this allows for the enrichment (via FACS) of reporter-positive cells, but do not inform which lipid nanoparticles (ENPs) facilitated that ‘functional delivery’ in the common case where multiple ENPs distribute to the same cell population. Thus, previous lipid nanoparticle (ENP) barcoded screening methods cannot distinguish between LNPs that enter the cell, but do not escape the endosome, and LNPs that escape the endosome into the cytosol. This is an important distinction when delivering polynucleotide payloads using an LNP because the payload is only transcribed and / or translated when delivered to the cytosol. Thus, while previous LNPs screening methods may identify LNPs that localize to a target cell, these methods poorly discriminate between LNPs that result in expression of the payload (e.g., a therapeutic) inside the cell (FIG. 1). For example, in FIG. 1 current LNP barcoding methods would indicate that LNPs A, B, C, and D successfully localized to the target cell type, despite only LNP D entering the cytosol. To overcome this problem, a self-amplifying RNA (saRNA) / trans-amplifying RNA (taRNA) LNP screening method has been developed to distinguish between LNP localization to a specific cell and the LNP entering the cytosol. In this method, LNPs comprise an saRNA or taRNA with a barcode. Each saRNA and taRNA comprises a polynucleotide comprising a nucleic acid encoding a replicase; and a polynucleotide comprising a nucleic acid comprising a barcode, operably linked to a conserved sequence element (CSE) cognate to the replicase, such that the barcode can be amplified by the replicase (e.g., as shown in FIG. 6). In taRNAs, the nucleic acids encoding the replicase and comprising the barcode are on separate polyribonucleotides (i.e., are not comprised in the same polyribonucleotide molecule). In saRNAs, the nucleic acids encoding the replicase and comprising the barcode are on the same polyribonucleotide (i.e., are comprised in the same polyribonucleotide molecule). According to this method, the RNA barcode comprised in the

[0240] 12015157.1 RNA cargo (i.e., in the saRNA and / or taRNA) is indicative of the type of LNP being used to deliver the same. If a given LNP enters the cytosol, the replicase encoded by the RNA cargo (i.e., encoded by the saRNA and / or the taRNA) will be produced by the cell’s ribosome. In turn the replicase can amplify the polynucleotide comprising the barcode. The amount of barcode amplification can be measured using next-generation sequencing, and is indicative of how well the LNP delivered the saRNA or taRNA to the cytosol. Increased barcode amplification indicates increased entry of the LNP into the cytosol. Thus, this method can distinguish between LNPs do not enter the cytosol (e.g., LNPs A, B, and C in FIG. 1), and LNPs that do enter the cytosol (e.g., LNP D).

[0241] Initial experiments were performed using a trans-amplifying RNA in vitro cell culture. Each trans-amplifying RNA had two components: (1) a trans-replicating RNA (trRNA) having a Sindbis Virus (SINV) 5’ untranslated region (UTR) (SINV 5’ -UTR), a CSE that is recognized by the SINV replicase, a green fluorescent protein (GFP) payload, a barcode (e.g., a barcode of Table 2), and a SINV 3 ’-UTR (FIG. 2A);and (2) a replicase construct encoding the SINV replicase. The barcode was inserted between the pay load and the SINV 3 ’-UTR in the following sequence context: CTCTACAAATGATAATAG-RNA barcode-TCGAGGCGGCCGCCACGC (SEQ ID NO: 429) (e.g., as shown in Table 2). The skilled artisan will appreciate that the recitation of an “RNA barcode” in SEQ ID NO: 429 represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length. In some embodiments, SEQ ID NO: 429represents any sequence of nucleotides (e.g., “N”s) that is 6-20 nts in length and otherwise conforming to the definition of “RNA barcode” as used throughout the instant disclosure. The barcodes (and the sequence context in which the barcode was comprised) were tested to ensure they had minimal to no secondary structure, as secondary structures may affect amplification activity by a replicase. The barcodes were also tested to ensure they did not have a mammalian miRNA seed region, which could also affect expression of the trRNA (e.g., the barcode). Barcodes that did not pass these tests were not included. trRNA plus and minus replicase was transfected into BHK-21 and 3T3 cells. 66ng trRNA was transfected into cells (+ / - 660ng Replicase). Media was changed after 2 hrs. RNA was isolated after 24hrs. The amount of trRNA was measured by RT-qPCR. Results shows that trRNA abundance increased when the replicase was cotransfected in both cells lines (FIG. 2B).

[0242] Additional experiments showed a positive correlation between the amount of trRNA added to the cell initially and the amount of trRNA produced after amplification by the replicase. Specifically, 1 Ibp trRNAs (“amp-trRNA”) with different barcodes were pooled together at

[0243] 12015157.1 intentionally different proportions across a >150x range. Cells were transfected with this pool (all in the same polyplex) with and without replicase constructs. Media was changed after 2 hrs. Cells were imaged and GFP+ cells were seen at 24hrs (FIG. 3A). RNA was isolated after 24hrs. RT-qPCR showed 66x amplification in the with replicase condition (FIG. 3B). Isolated trRNA was sequenced, and compared to the pool transfected (i.e., the input). The data show a strong correlation between isolated RNA (+ / - replicase) and the amount of trRNA transfected (FIGs. 3C). RNA for each condition (+ / - replicase isolated from cells & input RNA that was transfected onto cells) was normalized to transcripts per million (TPM) then normalized to the relative abundance in the input. The data indicate that the replicase can amplify barcoded trRNA in proportion to the amount delivered, which is important to inferring LNP delivery to the cytoplasm of a cell.

[0244] Two different LNP screening methods are implemented to determine LNP delivery of trRNA to the cytosol.

[0245] In the first method, different LNPs (with different chemical compositions) are loaded with a trRNA and a replicase construct (FIG. 4). The barcodes on the trRNA are indicates of the type of LNP the trRNA is in. The different LNPs are administered to a non-human primate, mouse or rat. After administration, the different tissues of the animal are extracted and sequenced to identify barcode amount and to determine barcode amplification.

[0246] In a second method, a first set of different LNPs are loaded with the trRNA, but not the replicase construct (FIG. 5). A second set of LNPs are loaded with the replicase construct. The first set and the second set of LNP are administered to a non-human primate, mouse, or rate. After administration, the different tissues of the animal are extracted and sequenced to identify barcode amounts and to determine barcode amplification.

[0247] In a third method, a first set of different LNPs are loaded with barcoded trRNAs, but not the replicase construct and a second set of LNPs are loaded with the replicase construct. (FIG. 6). Each LNP individually undergoes various methods of characterization of physiochemical properties, and multiple NPs are pooled and administered into one or multiple species. After a certain timepoint, tissues and (optionally) specific cell-types are isolated and the barcoded taRNA is then measured by NGS. These NGS results are then related to the NP composition (e.g. chemistry & ratio of components) and characterization results. These data may inform hit / lead nomination or inform iterative screening efforts.

[0248] Example 2. Production and Validation of Barcoded taRNA

[0249] 12015157.1 Design of barcoded RNAs

[0250] 8nt barcode sequences were designed such that they had a Levenshtein distance of 3+ relative to any other barcode. These barcodes were further filtered to remove barcodes containing mammalian miRNA seed regions (utilizing the TargetScan database) and to remove sequences with significant predicted secondary structure (within the context of a barcoded amp- trRNA by including 20 nucleotides 5’ and 3’ to the inserted barcode) using ViennaRNA package. Table 2 provides selected barcodes (“Barcodes”), as well as amp-trRNA containing the same barcodes (“Barcodes (bolded) in Context”).

[0251] Production of barcoded RNAs

[0252] 8nt barcode sequences were then cloned into a parental trRNA sequence (in this case, 5’ SINV UTR / EGFP / Bsal site / 3’ SINV UTR, followed by 35nt PolyA tail and BspQI site; e.g., SEQ ID NO: 7) at the Bsal site using golden gate cloning methods. DNA was linearized using BspQI and digested for 1+ hours following by DNA isolation using Zymo Cleanup Kits. Barcoded trRNA were then produced by in vitro transcription using linearized DNA template with a standard IVT reaction mix. This reaction mixture was incubated for 2-3 hours (37°C), after which DNasel and DNasel buffer were added, and the mixture was incubated for an additional 30-minute period (37°C). After the full incubation period, RNA was isolated using NEB Monarch RNA Cleanup kits according to manufacturer instructions, then eluted into lOOpL RNase-free water. Total RNA produced (ng) was measured via Nanodrop for each construct. RNA integrity was assessed via denaturing gel electrophoresis or Agilent Fragment Analyzer. RNA was stored at -20°C or 80°C.

[0253] In vitro validation

[0254] 200,000 BHK21 cells were seeded per well of a 6 well plate. After 24hrs, a pool of 11 different barcoded constructs at intentionally different ratios spanning a >150x range of concentrations was created. Cells were transfected with 66ng of the pooled barcoded trRNAs with and without 660ng SFV Replicase mRNA using MessengerMax (ThermoFischer) in biological duplicate. Media was changed after 2hrs of inoculation. After 24hrs, cells were imaged for GFP expression (FIG. 3A) and more GFP-positive cells were observed in the conditions co-transfected with SFV replicase. At this point, total RNA was isolated using Qiagen RNeasy Plus Kit. RT-qPCR was conducted using a probe designed to the EGFP open reading frame and observed >70x more trRNA in the wells that also received replicase mRNA

[0255] 12015157.1 as compared to those that did not (3B). In combination, FIG. 3 A and FIG. 3B indicate that barcoded trRNAs are capable of being amplified.

[0256] The barcode regions within the isolated RNA were reverse transcribed using Superscript IV utilizing primers specific to the EGFP open reading frame (ORF) and SINV 3’ UTR. Targeted RNA-seq was then performed on this amplicon with the MiSeq platform (Azenta) generating 250bp paired-end reads. After quality control and deduplication, barcode counts were quantified in each sample with abundance in total RNA from the RNA isolation from with and without replicase conditions and from the input RNA pool which were then compared. In FIG. 3C, a strong correlation between the barcode relative abundance in the input barcoded trRNA pool (e.g., before administration) and the relative abundance in barcoded trRNA isolated from cells with and without replicase (R = 0.9 and 0.99, respectfully). These data indicate that barcoded trRNAs amplify proportionally to their abundance within cells. FIG. 12 also shows that multiple barcoded trRNAs can be synthesized and have repeatable, consistent activity in mammalian cells.

[0257] Example 2. LNP Screening in vivo

[0258] Lipid nanoparticle components were dissolved in 100% ethanol at specific molar ratio of lipid components. The nucleic acid cargo - comprising a replicase and barcoded amplifying trRNA were dissolved in 50mM Sodium Acetate, pH=5, resulting in a concentration of nucleic acid cargo of approximately 0.2mg / mL RNA. In some embodiments, the molar ratio of replicase mRNA to barcoded amplifying trRNA is between 100:1 and 1:100. LNPs are formulated with an intended Nitrogen-to-Phosphate (N / P) ratio of between 3:1 and 8:1 using microfluidic mixing (e.g. Precision Nanosystems NanoAssemblr Ignite or custom T-mix utilizing a 2:1 or 3: 1 ratio of aqueous to organic solvent and a flow rate of approximately 12mL / min. After formulation, LNPs are diluted in equal volumes 20mM Tris Buffered Saline (pH=7.4) and further buffer exchanged via dialysis using a multi- well dialysis device.

[0259] At this point, physicochemical properties of barcoded LNPs including diameter, polydispersity, pKa, and encapsulation efficiency were measured using field standard assays of dynamic light scattering, TNS assay, and Ribogreen assay, respectively. Barcoded LNPs deemed acceptable per physicochemical assays were pooled together and sterile filtered using 0.22um filters prior to dosing to rodents or non-human primates. Animals were sacrificed 24 hours after LNP pool administration and tissues were placed immediately into RNAlater solution. RNA was then isolated using Trizol. Targeted RNA-seq was then performed as previously described and barcoded RNA abundance was normalized to the abundance in the pre-injection input.

[0260] 12015157.1 FIGs. 7 A and 7B illustrate proof of concept for screening LNPs using barcoded taRNA in mice. In figure 7A, mice were administered intramuscularly with barcoded mRNA (an existing method for LNP screening) and barcoded taRNA. The abundance of barcoded RNA was quantified over a time course via qPCR. As much as 45x more barcoded taRNA was present at 24 hours, representing an increase in signal due to amplification of the cytosolic delivered trRNA. In figure 7B, cells were transfected with an intentionally varying amount of barcoded trRNA. Relative abundance of each barcode was measured via NGS before and after cotransfection with a replicase into mammalian cells. The results being correlated indicates that barcoded taRNA assays have a robust linear range.

[0261] FIGs. 9A-9C are screening data wherein 8 LNP compositions were formulated into formulation duplicates. These 16 LNPs were then administered into 8 mice. After barcode quantification, minimal technical (between formulation duplicates) and biological (between mice) was observed for each 8 compositions (FIG. 9A). Technical and biological variance is illustrated in FIG. 9B- 9C for two field-known LNPs.

[0262] FIGs. 10A-10B show the correlation between barcode results (enrichment ratio) and the performance of each LNP individually (measured via IVIS) for a test pool of 5 compositions after intramuscular administration (FIG. 10A) and intravenous administration to liver expression (FIG. 10B).

[0263] FIGs. 11A-1 IB show the impact of the addition of GalNAc targeting ligands to three LNP compositions. Both barcoding and individual expression (via IVIS) indicate that the addition of GalNAc does not increase liver activity (FIG. 11 A), but does decrease splenic activity (FIG. 11B).

[0264] 12015157.1

Claims

CLAIMSWhat is claimed is:

1. A method of determining in vivo amplification of a plurality of ribonucleic acid (RNA) polynucleotides in cells of a specific cell type of a subject, the method comprising:(i) obtaining the plurality of RNA polynucleotides, wherein each RNA polynucleotide of the plurality of RNA polynucleotides comprises a conserved sequence element (CSE) and an RNA barcode;(ii) administering RNA polynucleotides of the plurality of RNA polynucleotides to the subject;(iii) extracting, from the subject, cells of the specific cell type, wherein the cells of the specific cell type comprise a replicase that is cognate to the CSE; and(iv) determining in vivo amplification of the administered RNA polynucleotides of the plurality of RNA polynucleotides in the subject by detecting each RNA barcode in the extracted cell of the specific cell type.

2. The method of claim 1, wherein RNA polynucleotides of the plurality of RNA polynucleotides further comprises a nucleic acid encoding a payload.

3. The method of claim 1 or 2, wherein RNA polynucleotides of the plurality of RNA polynucleotides comprise:(a) a 5’ alphavirus untranslated region (UTR);(b) the CSE;(c) a nucleic acid payload or a nucleic acid encoding a payload;(d) the RNA barcode; and(e) a 3’ alphavirus UTR.

4. The method of any one of claims 1-3, wherein RNA polynucleotides of the plurality of RNA polynucleotides comprise, from 5’ to 3’:(a) a 5’ UTR comprising the CSE;(b) a nucleic acid payload or a nucleic acid encoding a payload;(c) the RNA barcode; and(d) a 3’ alphavirus UTR.12015157.

15. The method of any one of claims 1-3, wherein RNA polynucleotides of the plurality of RNA polynucleotides comprise, from 5’ to 3’:(a) a 5’ UTR comprising the CSE;(b) a nucleic acid payload or a nucleic acid encoding a payload; and(c) a 3’ alphavirus UTR comprising the RNA barcode.

6. The method of any one of claims 3-5, wherein the 5’ alphavirus UTR is a Venezuelan Equine Encephalitis Virus (VEEV) 5 ’-UTR, Semliki Forest Virus (SFV) 5 ’-UTR, Sindbis Virus (SINV) 5 ’-UTR, or Chikungunya Virus (CHIKV) 5 ’-UTR.

7. The method of any one of claims 3-6, wherein the 3’ alphavirus UTR is a VEEV 3’- UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR.

8. The method of any one of claims 2-7, wherein the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR.

9. The method of any one of claims 2-8, wherein the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR.

10. The method of any one of claims 3-9, wherein the RNA barcode is indicative of a mutation in the 5’ alphavirus UTR and / or the 3’ alphavirus UTR of a corresponding RNA polynucleotide.

11. The method of any one of claims 1-10, wherein the RNA barcode comprises at least 6 nucleotides.

12. The method of claim 11, wherein the RNA barcode consists of 8 nucleotides.

13. The method of claim 11 or claim 12, wherein the RNA barcode comprises the RNA barcode set forth in any one of REF ID NOs. 429-848.

14. The method of any one of claims 1-13, wherein the RNA barcode has minimal to no predicted secondary structure.12015157.

115. The method of any one of claims 1-14, wherein the RNA barcode does not comprise a mammalian microRNA (miRNA) seed region or a miRNA target site.

16. The method of any one of claims 2-15, wherein the payload comprises a reporter protein.

17. The method of claim 16, wherein the reporter protein is green fluorescent protein (GFP), enhanced GFP (EGFP), mCherry, mKate, anchored heavy chain variable domain (aVHH), nano luciferase (nanoLuc), firefly luciferase (Flue), secreted embryonic alkaline phosphatase (SEAP), or Cre recombinase.

18. The method of any one of claims 2-17, wherein the payload is not a reporter protein.

19. The method of any one of claims 2-15, wherein the pay load is a therapeutic polynucleotide, or the payload encodes a therapeutic polypeptide.

20. The method of any one of claims 2-15, wherein the payload is a nuclease.

21. The method of any one of claims 2-15, wherein the payload does not encode a protein.

22. The method of any one of claims 2-21, wherein each different RNA barcode sequence in the plurality of RNA polynucleotides is at least a Hamming distance of 3 from each other.

23. The method of any one of claims 1-22, wherein RNA polynucleotides of the plurality of RNA polynucleotides further comprise nucleic acids encoding the replicase that is cognate to the CSE.

24. The method of any one of claims 1-23, further comprising contacting, in vivo, cells of the specific cell type of the subject with a polyribonucleotide encoding the replicase that is cognate to the CSE.

25. The method of any one of claims 1-24, wherein the plurality of RNA polynucleotides comprises at least 10 different RNA polynucleotides.12015157.

126. The method of claim 25, wherein the at least 10 different RNA polynucleotides each comprise a different 5’ alphavirus UTRs and / or a different 3’ alphavirus UTRs.

27. The method of any one of claims 1-26, wherein obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of nanoparticles, wherein nanoparticles of the plurality of nanoparticles comprise an RNA polynucleotide of the plurality of RNA polynucleotides.

28. The method of claim 27, wherein at least one nanoparticle of the plurality of nanoparticles comprises a nucleic acid encoding the replicase that is cognate to the CSE.

29. The method of claim 27 or 28, wherein contacting cells of the specific cell type comprises contacting, in vivo, the cells of the specific cell type with the plurality of nanoparticles.

30. The method of claim 27, wherein obtaining the plurality of nanoparticles comprises obtaining at least two different pluralities of nanoparticles, wherein the different pluralities of nanoparticles comprise a different RNA barcode.

31. The method of any one of claims 27-30, wherein nanoparticles of the plurality of nanoparticles are lipid nanoparticles.

32. The method of claim 31, wherein the plurality of nanoparticles comprises a first plurality of lipid nanoparticles and a second plurality of lipid nanoparticles, wherein: the first plurality of lipid nanoparticles has a formulation comprising at least one different component relative to the second plurality of lipid nanoparticles, the first plurality of lipid nanoparticles comprises a first plurality of RNA polynucleotides comprising a first RNA barcode and the second plurality of lipid nanoparticles comprises a second plurality of RNA polynucleotides comprising a second RNA barcode, and wherein the first and second RNA barcodes are different.12015157.

133. The method of any one of claims 27-30, wherein the nanoparticles are polymeric nanoparticles.

34. The method of claim 31, wherein the plurality of nanoparticles comprises a first plurality of polymeric nanoparticles and a second plurality of polymeric nanoparticles, wherein: the first plurality of polymeric nanoparticles has a formulation comprising at least one different component relative to the second plurality of polymeric nanoparticles, the first plurality of polymeric nanoparticles comprises a first plurality of RNA polynucleotides comprising a first RNA barcode and the second plurality of polymeric nanoparticles comprises a second plurality of RNA polynucleotides comprising a second RNA barcode, and wherein the first and second RNA barcodes are different.

35. The method of any one of claims 1-34, further comprising contacting the cells of the specific cell type with a replicase nanoparticle, the replicase nanoparticle (a) comprising a polyribonucleotide encoding the replicase that is cognate to the CSE and (b) not comprising an RNA polynucleotide of the plurality of RNA polynucleotides.

36. The method of any one of claims 27-35, wherein a nanoparticle of the plurality of nanoparticles comprises a guide RNA.

37. The method of any one of claims 1-26, wherein obtaining the plurality of RNA polynucleotides comprises obtaining a plurality of viral vectors, wherein viral vectors of the plurality of viral vectors comprise an RNA polynucleotide of the plurality of RNA polynucleotides.

38. The method of claim 37, wherein at least one viral vector of the plurality of viral vectors comprises a nucleic acid encoding the replicase that is cognate to the CSE.

39. The method of claim 37 or 38, wherein viral vectors of the plurality of viral vectors comprise adeno-associated viral vectors, lentiviral vectors, or adenoviral vectors.12015157.

140. The method of any one of claims 37-39, wherein contacting cells of the specific cell type comprises contacting, in vivo, the cells of the specific cell type with the plurality of viral vectors.

41. The method of any one of claims 1-40, wherein cells of the specific cell type of the subject have been engineered to express the replicase that is cognate to the CSE.

42. The method of any one of claims 1-26, wherein the obtaining the plurality of RNA polynucleotides comprises synthesizing the plurality of RNA polynucleotides using in vitro transcription.

43. The method of any one of claims 1-42, wherein contacting cells of the specific cell type comprises administering the plurality of RNA polynucleotides to the subject.

44. The method of any one of claims 1-43, wherein extracting cells of the specific cell type comprises extracting a biological sample that comprises the specific cell type from the subject.

45. The method of claim 44, further comprising isolating cells of the specific cell type from the biological sample using fluorescent assisted or magnetic cell sorting.

46. The method of any one of claims 1-44, wherein determining in vivo amplification of the plurality of RNA polynucleotides comprises sequencing the RNA barcodes of the RNA polynucleotides of the plurality of RNA polynucleotides to produce sequencing data.

47. The method of claim 46, wherein determining in vivo amplification of a selected RNA polynucleotide of the plurality of RNA polynucleotides in the cells of the specific cell type, comprises detecting the RNA barcode corresponding to the selected RNA polynucleotide in the cells of the specific cell type.

48. The method of claim 46, wherein determining in vivo amplification of a selected RNA polynucleotide of the plurality of RNA polynucleotides in the cell of the specific cell type, comprises using the sequencing data to measure an amount of the RNA barcode corresponding to the selected RNA polynucleotide in the cells of the specific cell type.12015157.

149. The method of any one of claims 1-48, wherein determining in vivo amplification of the plurality of RNA polynucleotides in the cells of the specific cell type comprises measuring an amount of the RNA barcode in the cells of the specific cell type relative to an amount of the RNA barcode in at least one other cell type of the subject.

50. A ribonucleic acid (RNA) polynucleotide comprising a 5’ alphavirus untranslated region (UTR), a conserved sequence element (CSE); an RNA barcode comprising at least 6 RNA nucleotides; and a 3’ alphavirus UTR.

51. The RNA polynucleotide of claim 50, wherein the 5’ alphavirus UTR comprises the CSE.

52. The RNA polynucleotide of claim 50 or 51, wherein the 3’ alphavirus UTR comprises the RNA barcode.

53. The RNA polynucleotide of any one of claims 50-52, wherein the 5’ alphavirus UTR is a Venezuelan Equine Encephalitis Virus (VEEV) 5’-UTR, Semliki Forest Virus (SFV) 5’-UTR, Sindbis Virus (SINV) 5 ’-UTR, or Chikungunya Virus (CHIKV) 5 ’-UTR.

54. The RNA polynucleotide of any one of claims 50-53, wherein the 3’ alphavirus UTR is a VEEV 3 ’-UTR, SFV 3 ’-UTR, SINV 3 ’-UTR, or CHIKV 3 ’-UTR.

55. The RNA polynucleotide of any one of claims 50-54, wherein the 5’ alphavirus UTR comprises a mutation relative to a wildtype 5’ alphavirus UTR.

56. The RNA polynucleotide of any one of claims 50-55, wherein the 3’ alphavirus UTR comprises a mutation relative to a wildtype 3’ alphavirus UTR.

57. The RNA polynucleotide of claim 55 or 56, wherein the RNA barcode is indicative of mutation in the 5’ alphavirus UTR and / or the 3’ alphavirus UTR.12015157.

158. The RNA polynucleotide of any one of claims 50-57, wherein each barcode has minimal to no predicted secondary structure.

59. The RNA polynucleotide of any one of claims 50-58, wherein each barcode does not comprise a mammalian microRNA (miRNA) seed region or target site.

60. The RNA polynucleotide of any one of claims 50-52, wherein the RNA barcode consists of 8 nucleotides.

61. The RNA polynucleotide of claim 53, wherein the RNA barcode is the RNA barcode set forth in any one of REF ID NOs.: 429-848.

62. The RNA polynucleotide of any one of claims 50-61, comprising the sequence set forth in any one of SEQ ID NOs: 9-428.

63. The RNA polynucleotide of any one of claims 50-62, wherein the RNA polynucleotide further comprises a nucleic acid payload or a nucleic acid encoding a payload.

64. The RNA polynucleotide of claim 63, wherein the payload comprises a reporter protein.

65. The RNA polynucleotide of claim 64, wherein the reporter protein is green fluorescent protein (GFP), enhanced GFP (EGFP), mCherry, mKate, anchored heavy chain variable domain (aVHH), nano luciferase (nanoLuc), firefly luciferase (Flue), Secreted embryonic alkaline phosphatase (SEAP), or Cre recombinase.

66. The RNA polynucleotide of claim 63, wherein the payload is not a reporter protein.

67. The RNA polynucleotide of claim 63 or 66, wherein the pay load is a therapeutic polynucleotide, or the payload encodes a therapeutic polypeptide.

68. The RNA polynucleotide of claim 63 or 66, wherein the pay load is a nuclease.12015157.

169. The RNA polynucleotide of claim 63 or 66, wherein the payload does not encode a protein.

70. The RNA polynucleotide of any one of claims 50-69, further comprising a nucleic acid encoding a replicase that is cognate to the CSE.

71. A trans-amplifying RNA comprising:(a) a first RNA polynucleotide comprising the RNA polynucleotide of any one of claims 50-69; and(b) a second RNA polynucleotide comprising a nucleic acid encoding a replicase that is cognate to the CSE.

72. A composition comprising a plurality of nanoparticles, wherein each nanoparticle of the plurality comprises the RNA polynucleotide of any one of claims 50-70 or the taRNA of claim 71.

73. A composition comprising a plurality of nanoparticles and a plurality of trans-amplifying ribonucleic acids (RNA) (taRNAs), wherein taRNAs of the plurality of taRNAs comprise: a first RNA polynucleotide comprising a conserved sequence element (CSE) and an RNA barcode; and a second RNA polynucleotide comprising a nucleic acid encoding a replicase that is cognate to the CSE; wherein the plurality of taRNAs comprises a first taRNA and second taRNA, wherein the first taRNA comprises a different RNA barcode relative to the second taRNA; and wherein the plurality of nanoparticles comprises a first plurality of nanoparticles and a second plurality of nanoparticles, wherein nanoparticles of the first plurality of nanoparticles comprises the first taRNA and do not comprise the second taRNA; and wherein nanoparticles of the second plurality of nanoparticles comprise the second taRNA and do not comprise the first taRNA.

74. A composition comprising a plurality of nanoparticles and a plurality of self- amplifying ribonucleic acids (RNA) (saRNAs),12015157.1wherein saRNAs of the plurality of saRNAs comprise an RNA polynucleotide comprising a conserved sequence element (CSE), an RNA barcode, and a nucleic acid encoding a replicase that is cognate to the CSE; wherein the plurality of saRNAs comprises a first saRNA and second saRNA, wherein the first saRNA comprises a different RNA barcode relative to the second saRNA; and wherein the plurality of nanoparticles comprises a first plurality of nanoparticles and a second plurality of nanoparticles, wherein nanoparticles of the first plurality of nanoparticles comprises the first saRNA and do not comprise the second saRNA; and wherein nanoparticles of the second plurality of nanoparticles comprise the second saRNA and do not comprise the first saRNA.

75. The composition of claim 73 or 74, wherein the first plurality of nanoparticles has a different formulation relative to the second plurality of nanoparticles.

76. A method of determining in vivo amplification of a plurality of RNA polynucleotides in a specific cell type of a subject, the method comprising:(i) obtaining plurality of nanoparticles collectively comprising a plurality of RNA polynucleotides, wherein each RNA polynucleotide of the plurality of RNA polynucleotides comprises a conserved sequence element (CSE) and an RNA barcode;(ii) contacting, in vivo, nanoparticles of the plurality of nanoparticles with cells of the specific cell type of the subject;(iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE; and(iv) determining in vivo amplification of the plurality of RNA polynucleotides in the subject by measuring an amount of each RNA barcode in the extracted cells of the specific cell type.

77. A method of determining delivery of a plurality of RNA polynucleotides to the cytosol of a specific cell type of a subject, the method comprising:(i) obtaining a plurality of nanoparticles collectively comprising a plurality of RNA polynucleotides, wherein each RNA polynucleotide of the plurality of RNA polynucleotides comprises a conserved sequence element (CSE) and an RNA barcode;12015157.1(ii) contacting, in vivo, nanoparticles of the plurality of nanoparticles with cells of the specific cell type of the subject;(iii) extracting, from the subject, cells of the specific cell type, wherein cells of the specific cell type comprise a replicase that is cognate to the CSE;(iv) determining delivery of the plurality of RNA polynucleotides to the cytosol of the specific cell type by measuring an amount of RNA barcode produced by in vivo amplification in the cytosol of the extracted cells of the specific cell type.

78. The method of claim 77, wherein the plurality of nanoparticles comprises lipid nanoparticles.

79. A method of preparing a composition for delivery of ribonucleic acid (RNA) cargo to the cytosol of a cell of a specific cell type in a subject, the method comprising:(a) obtaining a first plurality of nanoparticles and a second plurality of nanoparticles, wherein: the first plurality of nanoparticles has a different formulation relative to the second plurality of nanoparticles, the first plurality of nanoparticles comprises a first RNA polynucleotide comprising a first RNA barcode and the second plurality of nanoparticles comprises a second RNA polynucleotide comprising a second RNA barcode, and wherein the first and second RNA barcodes are different;(b) administering the first and second pluralities of nanoparticles to the subject;(c) determining that at least one of the first and / or second RNA polynucleotides was delivered to the cytosol of cells of the specific cell type by identifying the RNA barcode present in the cytosol of the cells of the specific cell type; and(d) preparing a composition comprising a plurality of nanoparticles comprising RNA cargo and having the formulation of the nanoparticle comprising the RNA barcode identified as delivered to the cytosol of the cells of the specific cell type in step (c).

80. The method of claim 79, wherein the first plurality of nanoparticles and the second plurality of nanoparticles are a first plurality of lipid nanoparticles and a second plurality of lipid nanoparticles.12015157.1

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